Cells immortalized with telomerase reverse transcriptase for use in drug screening

ABSTRACT

The invention provides compositions and methods related to human telomerase reverse transcriptase (hTRT), the catalytic protein subunit of human telomerase. The polynucleotides and polypeptides of the invention are useful for diagnosis, prognosis and treatment of human diseases, for changing the proliferative capacity of cells and organisms, and for identification and screening of compounds and treatments useful for treatment of diseases such as cancers.

This application is a continuation of U.S. patent application Ser. No.08/974,549 filed Nov. 19, 1997, U.S. Pat. No. 6,166,178, which is acontinuation-in-part of U.S. patent application Ser. No. 08/915,503,filed Aug. 14, 1997, abandoned, which is a continuation-in-part of U.S.patent application Ser. No. 08/912,951, filed Aug. 14, 1997, U.S. Pat.No. 6,475,789 and a continuation-in-part of U.S. patent application Ser.No. 08/911,312, filed Aug. 14, 1997, abandoned, all three of which arecontinuations-in-part of U.S. patent application Ser. No. 08/854,050,filed May 9, 1997, U.S. Pat. No. 6,261,836, which is acontinuation-in-part of U.S. patent application Ser. No. 08/851,843,filed May 6, 1997, U.S. Pat. No. 6,093,809, which is acontinuation-in-part of U.S. patent application Ser. No. 08/846,017,filed Apr. 25, 1997, abandoned. This application also claims priority toPatent Convention Treaty Patent Application Serial No.: PCT/US97/17885(published on Apr. 9, 1998 as WO 98/14593) and to Patent ConventionTreaty Patent Application Serial No. PCT/US97/17618 (published on Apr.9, 1998 as WO98/14592), both designating the U.S. and filed in the U.S.Receiving Office on Oct. 1, 1997. Each of the aforementionedapplications is explicitly incorporated herein by reference in itsentirety and for all purposes, This application also incorporates byreference copending U.S. patent application Ser. No. 08/974,584, filedNov. 19, 1997, in its entirety and for all purposes.

This invention was made with Government support under Grant No. GM28039,awarded by the National Institutes of Health. The Government has certainrights in this invention.

FIELD OF THE INVENTION

The present invention is related to novel nucleic acids encoding thecatalytic subunit of telomerase and related polypeptides. In particular,the present invention is directed to the catalytic subunit of humantelomerase. The invention provides methods and compositions relating tomedicine, molecular biology, chemistry, pharmacology, and medicaldiagnostic and prognostic technology.

BACKGROUND OF THE INVENTION

The following discussion is intended to introduce the field of thepresent invention to the reader. The citation of various references inthis section is not to be construed as an admission of prior invention.

It has long been recognized that complete replication of the ends ofeukaryotic chromosomes requires specialized cell components (Watson,1972, Nature New Biol., 239:197; Olovnikov, 1973, J. Theor. Biol.,41:181). Replication of a linear DNA strand by conventional DNApolymerases requires an RNA primer, and can proceed only 5′ to 3′. Whenthe RNA bound at the extreme 5′ ends of eukaryotic chromosomal DNAstrands is removed, a gap is introduced, leading to a progressiveshortening of daughter strands with each round of replication. Thisshortening of telomeres, the protein-DNA structures physically locatedon the ends of chromosomes, is thought to account for the phenomenon ofcellular senescence or aging (see, e.g., Goldstein, 1990, Science249:1129; Martin et al., 1979, Lab. Invest. 23:86; Goldstein et al.,1969, Proc. Natl. Acad. Sci. USA 64:155; and Schneider and Mitsui, 1976,Proc. Natl. Acad. Sci. USA, 73:3584) of normal human somatic cells invitro and in vivo.

The length and integrity of telomeres is thus related to entry of a cellinto a senescent stage (i.e., loss of proliferative capacity). Moreover,the ability of a cell to maintain (or increase) telomere length mayallow a cell to escape senescence, i.e., to become immortal.

The structure of telomeres and telomeric DNA has been investigated innumerous systems (see, e.g, Harley and Villeponteau, 1995, Curr. Opin.Genet. Dev. 5:249). In most organisms, telomeric DNA consists of atandem array of very simple sequences; in humans and other vertebratestelomeric DNA consists of hundreds to thousands of tandem repeats of thesequence TTAGGG. Methods for determining and modulating telomere lengthin cells are described in PCT Publications WO 93/23572 and WO 96/41016.

The maintenance of telomeres is a function of a telomere-specific DNApolymerase known as telomerase. Telomerase is a ribonucleoprotein (RNP)that uses a portion of its RNA moiety as a template for telomere repeatDNA synthesis (Morin, 1997, Eur. J. Cancer 33:750; Yu et al., 1990,Nature 344:126; Singer and Gottschling, 1994, Science 266:404; Autexierand Greider, 1994, Genes Develop., 8:563; Gilley et al., 1995, GenesDevelop., 9:2214; McEachern and Blackburn, 1995, Nature 367:403;Blackburn, 1992, Ann. Rev. Biochem., 61:113; Greider, 1996, Ann. Rev.Biochem., 65:337). The RNA components of human and other telomeraseshave been cloned and characterized (see, PCT Publication WO 96/01835 andFeng et al., 1995, Science 269:1236). However, the characterization ofthe protein components of telomerase has been difficult. In part, thisis because it has proved difficult to purify the telomerase RNP, whichis present in extremely low levels in cells in which it is expressed.For example, it has been estimated that human cells known to expresshigh levels of telomerase activity may have only about one hundredmolecules of the enzyme per cell.

Consistent with the relationship of telomeres and telomerase to theproliferative capacity of a cell (i.e., the ability of the cell todivide indefinitely), telomerase activity is detected in immortal celllines and an extraordinarily diverse set of tumor tissues, but is notdetected (i.e., was absent or below the assay threshold) in normalsomatic cell cultures or normal tissues adjacent to a tumor (see, U.S.Pat. Nos. 5,629,154; 5,489,508; 5,648,215; and 5,639,613; see also,Morin, 1989, Cell 59: 521; Shay and Bacchetti 1997, Eur. J. Cancer33:787; Kim et al., 1994, Science 266:2011; Counter et al., 1992, EMBOJ. 11:1921; Counter et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91,2900; Counter et al., 1994, J. Virol. 68:3410). Moreover, a correlationbetween the level of telomerase activity in a tumor and the likelyclinical outcome of the patient has been reported (e.g., U.S. Pat. No.5,639,613, supra, Langford et al., 1997, Hum. Pathol. 28:416).Telomerase activity has also been detected in human germ cells,proliferating stem or progenitor cells, and activated lymphocytes. Insomatic stem or progenitor cells, and in activated lymphocytes,telomerase activity is typically either very low or only transientlyexpressed (see, Chiu et al., 1996, Stem Cells 14:239; Bodnar et al.,1996, Exp. Cell Res. 228:58; Taylor et al., 1996, J. Invest. Dermatology106: 759).

Human telomerase is an ideal target for diagnosing and treating humandiseases relating to cellular proliferation and senescence, such ascancer. Methods for diagnosing and treating cancer and othertelomerase-related diseases in humans are described in U.S. Pat. Nos.5,489,508, 5,639,613, and 5,645,986. Methods for predicting tumorprogression by monitoring telomerase are described in U.S. Pat. No.5,639,613. The discovery and characterization of the catalytic proteinsubunit of human telomerase would provide additional useful assays fortelomerase and for disease diagnosis and therapy. Moreover, cloning anddetermination of the primary sequence of the catalytic protein subunitwould allow more effective therapies for human cancers and otherdiseases related to cell proliferative capacity and senescence.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an isolated, substantially pure, orrecombinant protein preparation of a telomerase reverse transcriptaseprotein, or a variant thereof, or a fragment thereof. In one embodimentthe protein is characterized as having a defined motif that has an aminoacid sequence:

Trp-R₁-X₇-R₁-R₁-R₂-X-Phe-Phe-Tyr-X-Thr-Glu-X₈₋₉-R₃-R₃-Arg-R₄-X₂-Trp (SEQID NOS:11 and 12)

where X is any amino acid and a subscript refers to the number ofconsecutive residues, R₁ is leucine or isoleucine, R₂ is glutamine orarginine, R₃ is phenylalanine or tyrosine, and R₄ is lysine orhistidine. In one embodiment the protein has a sequence of human TRT. Inother embodiments, the invention relates to peptides and polypeptidessharing substantial sequence identity with a subsequence of suchproteins.

In a related embodiment the invention provides an isolated,substantially pure or recombinant nucleic acid that encodes a telomerasereverse transcriptase protein. In one embodiment the nucleic acidencodes a protein comprising an amino acid sequence (SEQ ID NOS:11 and12):

Trp-R₁-X₇-R₁-R₁-R₂-X-Phe-Phe-Tyr-X-Thr-Glu-X₈₋₉-R₃-R₃-Arg-R₄-X₂-Trp.

In another embodiment, the nucleic acid has a sequence that encodes thehuman TRT protein. In other embodiments, the invention relates tooligonucleotides and polynucleotides sharing substantial sequenceidentity or complementarity with a subsequence of such nucleic acids.

In one embodiment, the invention relates to human telomerase reversetranscriptase (hTRT) protein. Thus, in one embodiment, the inventionprovides an isolated, substantially pure, or recombinant proteinpreparation of an hTRT protein, or a variant thereof, or a fragmentthereof. In one embodiment, the protein is characterized by having anamino acid sequence with at least about 75% or at least about 80%sequence identity to the hTRT protein of FIG. 17 (SEQ ID NO:2), or avariant thereof, or a fragment thereof. In a related aspect, the hTRTprotein has the sequence of SEQ ID NO:2. In some embodiments, theprotein has one or more telomerase activities, such as catalyticactivity. In one embodiment, the hTRT protein fragment has at least 6amino acid residues. In other embodiments, the hTRT protein fragment hasat least 8, at least about 10, at least about 12, at least about 15 orat least about 20 contiguous amino acid residues of a naturallyoccurring hTRT polypeptide. In still other embodiments, the hTRT proteinfragment has at least about 50 or at least about 100 amino acidresidues.

The invention also provides a composition comprising an hTRT protein andan RNA. The RNA may be a telomerase RNA, such as a human telomerase RNA.In one embodiment, the hTRT protein and the human telomerase RNA (hTR)form a ribonucleoprotein complex with a telomerase activity.

In one embodiment, the invention provides isolated human telomerasecomprising hTRT protein, such as a substantially pure human telomerasecomprising hTRT protein and comprising hTR. In one embodiment, thetelomerase is at least about 95% pure. The telomerase may be isolatedfrom a cell, such as a recombinant host cell in or a cell that expressestelomerase activity.

In another aspect, the invention provides an isolated, synthetic,substantially pure, or recombinant polynucleotide comprising a nucleicacid sequence that encodes an hTRT protein. In one embodiment, thepolynucleotide has a nucleotide sequence encoding an hTRT protein thathas an amino acid sequence as set forth in FIG. 17 (SEQ ID NO:2) or asequence that comprises one or more conservative amino acid (or codon)substitutions or one or more activity-altering amino acid (or codon)substitutions in said amino acid sequence. In a related aspect, thepolynucleotide hybridizes under stringent conditions to a polynucleotidehaving the sequence as set forth in FIG. 16 (SEQ ID NO:1). In anotherrelated aspect, the nucleotide sequence of the polynucleotide has asmallest sum probability of less than about 0.5 when compared to anucleotide sequence as set forth in FIG. 16 (SEQ ID NO:1) using BLASTalgorithm with default parameters.

In another aspect, the invention provides a polynucleotide having apromoter sequence operably linked to the sequence encoding the hTRTprotein. The promoter may be a promoter other than the naturallyoccurring hTRT promoter. In a related aspect, the invention provides anexpression vector comprising the promoter of the hTRT.

The invention also provides an isolated, synthetic, substantially pure,or recombinant polynucleotide that is at least ten nucleotides in lengthand comprises a contiguous sequence of at least ten nucleotides that isidentical or exactly complementary to a contiguous sequence in anaturally occurring hTRT gene or hTRT mRNA. In some embodiments thepolynucleotide is an RNA, a DNA, or contains one or more non-naturallyoccurring, synthetic nucleotides. In one aspect, the polynucleotide isidentical or exactly complementary to the contiguous sequence of atleast ten contiguous nucleotides in a naturally occurring hTRT gene orhTRT mRNA. For example, the polynucleotide may be an antisensepolynucleotide. In one embodiment, the antisense polynucleotidecomprises at least about 20 nucleotides.

The invention further provides a method of preparing recombinanttelomerase by contacting a recombinant hTRT protein with a telomeraseRNA component under conditions such that said recombinant protein andsaid telomerase RNA component associate to form a telomerase enzymecapable of catalyzing the addition of nucleotides to a telomerasesubstrate. In one embodiment, the hTRT protein has a sequence as setforth in FIG. 17 (SEQ ID NO:2). The hTRT protein may be produced in anin vitro expression system and mixed with a telomerase RNA or, inanother embodiment, the telomerase RNA can be co-expressed in the invitro expression system. In one embodiment the telomerase RNA is hTR. Inan alternative embodiment, the contacting occurs in a cell, such as ahuman cell. In one embodiment, the cell does not have telomeraseactivity prior to the contacting of the hTRT and the RNA, or theintroduction, such as by transfection, of an hTRT polynucleotide. In oneembodiment, the telomerase RNA is expressed naturally by said cell.

The invention also provides a cell, such as a human, mouse, or yeastcell, containing the recombinant polynucleotides of the invention suchas a polynucleotide with an hTRT protein coding sequence operably linkeda promoter. In particular aspects, the cell is a vertebrate cell, suchas a cell from a mammal, for example a human, and has an increasedproliferative capacity relative to a cell that is otherwise identicalbut does not comprise the recombinant polynucleotide or has an increasedtelomerase activity level relative to a cell that is otherwise identicalbut does not comprise the recombinant polynucleotide. In someembodiments the cell is immortal.

In related embodiments, the invention provides organisms and cellscomprising a polynucleotide encoding a human telomerase reversetranscriptase polypeptide, such as a transgenic non-human organism suchas a yeast, plant, bacterium, or a non-human animal, for example, amouse. The invention also provides for transgenic animals and cells fromwhich an hTRT gene has been deleted (knocked-out) or mutated such thatthe gene does not express a naturally occurring hTRT gene product. Thus,in alternative embodiments, the transgenic non-human animal has amutated telomerase gene, is an animal deficient in a telomeraseactivity, is an animal whose TRT deficiency is a result of a mutatedgene encoding a TRT having a reduced level of a telomerase activitycompared to a wild-type TRT and is an animal having a mutated TRT genewith one or more mutations, including missense mutations nonsensemutations, insertions, or deletions.

The invention also provides an isolated or recombinant antibody, orfragment thereof, that specifically binds to an hTRT protein. In oneembodiment, the antibody binds with an affinity of at least about 10⁸M⁻¹. The antibody may be monoclonal or may be a polyclonal composition,such as a polyclonal antisera. In a related aspect, the inventionprovides a cell capable of secreting the antibody, such as a hybridoma.

The invention also provides a method for determining whether a compoundor treatment is a modulator of a telomerase reverse transcriptaseactivity or hTRT expression. The method involves detecting or monitoringa change in activity or expression in a cell, animal or compositioncomprising an hTRT protein or polynucleotide following administration ofthe compound or treatment. In one embodiment, the method includes thesteps of providing a TRT composition, contacting the TRT with the testcompound and measuring the activity of the TRT, where a change in TRTactivity in the presence of the test compound is an indicator that thetest compound modulates TRT activity. In certain embodiments, thecomposition is a cell, an organism, a transgenic organism or an in vitrosystem, such as an expression system, which contains a recombinantpolynucleotide encoding an hTRT polypeptide. Thus, the hTRT of themethod may be a product of in vitro expression. In various embodimentsthe detection of telomerase activity or expression may be by detecting achange in abundance of an hTRT gene product, monitoring incorporation ofa nucleotide label into a substrate for telomerase, monitoringhybridization of a probe to an extended telomerase substrate, monitoringamplification of an extended telomerase substrate, monitoring telomerelength of a cell exposed to the test compound, monitoring the loss ofthe ability of the telomerase to bind to a chromosome, or measuring theaccumulation or loss of telomere structure.

In one aspect, the invention provides a method of detecting an hTRT geneproduct in a biological sample by contacting the biological sample witha probe that specifically binds the gene product, wherein the probe andthe gene product form a complex, and detecting the complex, where thepresence of the complex is correlated with the presence of the hTRT geneproduct in the biological sample. The gene product may be RNA, DNA or apolypeptide. Examples of probes that may be used for detection include,but are not limited to, nucleic acids and antibodies.

In one embodiment, the gene product is a nucleic acid which is detectedby amplifying the gene and detecting the amplification product, wherethe presence of the complex or amplification product is correlated withthe presence of the hTRT gene product in the biological sample.

In one embodiment, the biological sample is from a patient, such as ahuman patient. In another embodiment the biological sample includes atleast one cell from an in vitro cell culture, such as a human cellculture.

The invention further provides a method of detecting the presence of atleast one immortal or telomerase positive human cell in a biologicalsample comprising human cells by obtaining the biological samplecomprising human cells; and detecting the presence in the sample of acell having a high level of an hTRT gene product, where the presence ofa cell having a high level of the hTRT gene product is correlated withthe presence of immortal or telomerase positive cells in the biologicalsample.

The invention also provides a method for diagnosing a telomerase-relatedcondition in a patient by obtaining a cell or tissue sample from thepatient, determining the amount of an hTRT gene product in the cell ortissue; and comparing the amount of hTRT gene product in the cell ortissue with the amount in a healthy cell or tissue of the same type,where a different amount of hTRT gene product in the sample from thepatient and the healthy cell or tissue is diagnostic of atelomerase-related condition. In one embodiment the telomerase-relatedcondition is cancer and a greater amount of hTRT gene product isdetected in the sample.

The invention further provides a method of diagnosing cancer in apatient by obtaining a biological sample from the patient, and detectinga hTRT gene product in the patient sample, where the detection of thehTRT gene product in the sample is correlated with a diagnosis ofcancer.

The invention further provides a method of diagnosing cancer in apatient by obtaining a patient sample, determining the amount of hTRTgene product in the patient sample; and comparing the amount of hTRTgene product with a normal or control value, where an amount of the hTRTgene product in the patient that is greater than the normal or controlvalue is diagnostic of cancer.

The invention also provides a method of diagnosing cancer in a patient,by obtaining a patient sample containing at least one cell; determiningthe amount of an hTRT gene product in a cell in the sample; andcomparing the amount of hTRT gene product in the cell with a normalvalue for the cell, wherein an amount of the hTRT gene product greaterthan the normal value is diagnostic of cancer. In one embodiment, thesample is believed to contain at least one malignant cell.

The invention also provides a method for a prognosing a cancer patientby determining the amount of hTRT gene product in a cancer cell obtainedfrom the patient; and comparing the amount of hTRT in the cancer cellwith a prognostic value of hTRT consistent with a prognosis for thecancer; where an amount of hTRT in the sample that is at the prognosticvalue provides the particular prognosis.

The invention also provides a method for monitoring the ability of ananticancer treatment to reduce the proliferative capacity of cancercells in a patient, by making a first measurement of the amount of anhTRT gene product in at least one cancer cell from the patient; making asecond measurement of the level of the hTRT gene product in at least onecancer cell from the patient, wherein the anticancer treatment isadministered to the patient before the second measurement; and comparingthe first and second measurements, where a lower level of the hTRT geneproduct in the second measurement is correlated with the ability of ananticancer treatment to reduce the proliferative capacity of cancercells in the patient.

The invention also provides kits for the detection of an hTRT gene orgene product. In one embodiment, the kit includes a container includinga molecule selected from an hTRT nucleic acid or subsequence thereof, anhTRT polypeptide or subsequence thereof, and an anti-hTRT antibody.

The invention also provides methods of treating human diseases. In oneembodiment, the invention provides a method for increasing theproliferative capacity of a vertebrate cell, such as a mammalian cell,by introducing a recombinant polynucleotide into the cell, wherein saidpolynucleotide comprises a sequence encoding an hTRT polypeptide. In oneembodiment, the hTRT polypeptide has a sequence as shown in FIG. 17. Inone embodiment, the sequence is operably linked to a promoter. In oneembodiment, the hTRT has telomerase catalytic activity. In oneembodiment, the cell is human, such as a cell in a human patient. In analternative embodiment, the cell is cultured in vitro. In a relatedembodiment, the cell is introduced into a human patient.

The invention further provides a method for treating a human disease byintroducing recombinant hTRT polynucleotide into at least one cell in apatient. In one embodiment, a gene therapy vector is used. In a relatedembodiment, the method further consists of introducing into the cell apolynucleotide comprising a sequence encoding hTR, for example, an hTRpolynucleotide operably linked to a promoter.

The invention also provides a method for increasing the proliferativecapacity of a vertebrate cell, said method comprising introducing intothe cell an effective amount of hTRT polypeptide. In one embodiment thehTRT polypeptide has telomerase catalytic activity. The inventionfurther provides cells and cell progeny with increased proliferativecapacity.

The invention also provides a method for treating a condition associatedwith an elevated level of telomerase activity within a cell, comprisingintroducing into said cell a therapeutically effective amount of aninhibitor of said telomerase activity, wherein said inhibitor is an hTRTpolypeptide or an hTRT polynucleotide. In one embodiment, the inhibitoris a polypeptide or polynucleotide comprising, e.g., at least asubsequence of a sequence shown in FIGS. 16, 17, or 20. In additionalembodiments, the polypeptide or polynucleotide inhibits a TRT activity,such as binding of endogenous TRT to telomerase RNA.

The invention also provides a vaccine comprising an hTRT polypeptide andan adjuvant. The invention also provides pharmacological compositionscontaining a pharmaceutically acceptable carrier and a molecule selectedfrom: an hTRT polypeptide, a polynucleotide encoding an hTRTpolypeptide, and an hTRT nucleic acid or subsequence thereof.

DESCRIPTION OF THE FIGURES

FIG. 1 shows highly conserved residues in TRT motifs from human (SEQ IDNO:13), S. pombe (tez1) (SEQ ID NO:14), S. cerevisiae (EST2) (SEQ IDNO:15) and Euplotes aediculatus (p123) (SEQ ID NO:16). Identical aminoacids are indicated with an asterisk (*) [raised slightly], while thesimilar amino acid residues are indicated by a dot (•). Motif “0” in thefigure is also called Motif T; Motif “3” is also called Motif A.

FIG. 2 shows the location of telomerase-specific and RT-specificsequence motifs of telomerase proteins and other reverse transcriptases.Locations of telomerase-specific motif T and conserved RT motifs 1, 2and A-E are indicated by boxes. The open rectangle labeled HIV-1 RTdelineates the portion of this protein shown in FIG. 3.

FIG. 3 shows the crystal structure of the p66 subunit of HIV-1 reversetranscriptase (Brookhaven code 1HNV). The view is from the back of theright hand to enable all motifs to be shown.

FIG. 4 shows multiple sequence alignment of telomerase RTs (Sp_Trt1p, S.pombe TRT (SEQ ID NOS:24-29) [also referred to herein as “tez1p”]; hTRT,human TRT (SEQ ID NOS:30-35); Ea_p123, Euplotes p123 (SEQ ID NOS:36-41);Sc_Est2p, S. cerevisiae Est2p) (SEQ ID NOS:42-48) and members of otherRT families (Sc_al, cytochrome oxidase group II intron 1-encoded proteinfrom S. cerevisiae mitochondria (SEQ ID NOS:51-56), Dm_TART, reversetranscriptase from Drosophila melanogaster TART non-LTRretrotransposable element) (SEQ ID NOS:57-63; HIV-1, humanimmunodeficiency virus reverse transcriptase (SEQ ID NOS:64-68)). TRTcon (SEQ ID NOS:17-23) and RT con (SEQ ID NOS:49 and 50) representconsensus sequences for telomerase RTs and non-telomerase RTs. Aminoacids are designated with an h, hydrophobic; p, polar; c, charged.Triangles show residues that are conserved among telomerase proteins butdifferent in other RTs. The solid line below motif E highlights theprimer grip region.

FIG. 5 shows expression of hTRT RNA in telomerase-negative mortal cellstrains and telomerase-positive immortal cell lines as described inExample 2.

FIG. 6 shows a possible phylogenetic tree of telomerases andretroelements rooted with RNA-dependent RNA polymerases.

FIG. 7 shows a restriction map of lambda clone Gφ5.

FIG. 8 shows a map of chromosome 5p with the location of the STS markerD5S678 (located near the hTRT gene) indicated.

FIG. 9 shows the construction of a hTRT promoter-reporter plasmid.

FIGS. 10A and B, in two pages, shows coexpression in vitro of hTRT andhTR to produce catalytically active human telomerase.

FIG. 11, in two pages, shows an alignment of sequences from four TRTproteins from human (hTRT; SEQ ID NOS:72-79), S. pombe Trt1 (spTRT; SEQID NOS:80-87), Euplotes p123 (Ea_p123; SEQ ID NOS:88-95), and S.cerevisiae EST2p TRT (Sc_Est2; SEQ ID NOS:96-104) and identifies motifsof interest. TRT con (SEQ ID NOS:69, 21, 70 and 71) shows a TRTconsensus sequence. RT con (SEQ ID NOS:49 and 50) shows consensusresidues for other reverse transcriptases. Consensus residues in uppercase indicate absolute conservation in TRT proteins.

FIG. 12 shows a Topoisomerase II cleavage site (SEQ ID NO: 108) and NFkBbinding site motifs (NFkB_CS1=SEQ ID NO:105; NFkB-MHC-I.2=SEQ ID NO:106;NFkB_CS2=SEQ ID NO:107) in an hTRT intron, with the sequence showncorresponding to SEQ ID NO:7.

FIG. 13, in two pages, shows the sequence of the DNA encoding theEuplotes 123 kDa telomerase protein subunit (Euplotes TRT; SEQ IDNO:109).

FIG. 14 shows the amino acid sequence of the Euplotes 123 kDa telomeraseprotein subunit (Euplotes TRT protein; SEQ ID NO:110).

FIG. 15, in five pages, shows the DNA (SEQ ID NO:111) and amino acid(SEQ ID NO:112) sequences of the S. pombe telomerase catalytic subunit(S. pombe TRT).

FIG. 16, in two pages, shows the hTRT cDNA sequence, with the sequenceshown corresponding to SEQ ID NO:1.

FIG. 17 shows the hTRT protein encoded by the cDNA of FIG. 16. Theprotein sequence shown corresponds to SEQ ID NO:2.

FIG. 18 shows the sequence of clone 712562, with the sequence showncorresponding to SEQ ID NO:3.

FIG. 19 shows a 259 residue protein encoded by clone 712562, with thesequence shown corresponding to SEQ ID NO:10.

FIG. 20 shows, in seven pages, the sequence of a nucleic acid with anopen reading frame encoding a Δ182 variant polypeptide, with thesequence shown corresponding to SEQ ID NO:4. This Figure also shows theamino acid sequence of this Δ182 variant polypeptide, with the aminoacid sequence shown corresponding to SEQ ID NO:5.

FIG. 21 shows, in six pages, sequence from an hTRT genomic clone, withthe sequence shown corresponding to SEQ ID NO:6. Consensus motifs andelements are indicated, including sequences characteristic of atopoisomerase II cleavage site, NFκB binding sites, an Alu sequence. andother sequence elements.

FIGS. 22A, B, C, D shows the effect of mutation of the TRT gene inyeast, as described in Example 1.

FIG. 23 shows the sequence of EST AA281296, corresponding to SEQ IDNO:8.

FIG. 24 shows the sequence of the 182 basepairs deleted in clone 712562,with the sequence shown corresponding to SEQ ID NO:9.

FIG. 25 shows the results of an assay for telomerase activity from BJcells transfected with an expression vector encoding an hTRT protein(pGRN133) or a control plasmid (pBBS212) as described in Example 13.

FIGS. 26A and B is a schematic diagram of the affinity purification oftelomerase showing the binding and displacement elution steps.

FIG. 27 is a photograph of a Northern blot of telomerase preparationsobtained during a purification protocol, as described in Example 1. Lane1 contained 1.5 fmol telomerase RNA, lane 2 contained 4.6 fmoltelomerase RNA, lane 3 contained 14 fmol telomerase RNA, lane 4contained 41 fmol telomerase RNA, lane 5 contained nuclear extract (42fmol telomerase), lane 6 contained Affi-Gel-heparin-purified telomerase(47 fmol telomerase), lane 7 contained affinity-purified telomerase (68fmol), and lane 8 contained glycerol gradient-purified telomerase (35fmol).

FIG. 28 shows telomerase activity through a purification protocol.

FIG. 29 is a photograph of a SDS-PAGE gel, showing the presence of anapproximately 123 kDa polypeptide and an approximately 43 kDa doubletfrom Euplotes aediculatus.

FIG. 30 is a graph showing the sedimentation coefficient of Euplotesaediculatus telomerase.

FIG. 31 is a photograph of a polyacrylamide/urea gel with 36% formamideshowing the substrate utilization of Euplotes telomerase.

FIGS. 32A and B shows the putative alignments of telomerase RNA template(SEQ ID NO:113), and hairpin primers with telomerase RNA.

FIG. 33 is a photograph of lanes 25-30 of the gel shown in FIG. 31,shown at a lighter exposure level (G₄T₄G₄T₄=SEQ ID NO:114).

FIG. 34 shows the DNA sequence of the gene encoding the 43 kDatelomerase protein subunit from Euplotes (SEQ ID NO:115).

FIG. 35 shows, in four pages, the DNA sequence (SEQ ID NO:115), as wellas the amino acid sequences of all three open reading frames of the 43kDa telomerase protein subunit from Euplotes (a=SEQ ID NOS:116-140;b=SEQ ID NOS:141-162; c=SEQ ID NOS:163-186).

FIG. 36 shows a sequence comparison between the 123 kDa telomeraseprotein subunit of Euplotes (SEQ ID NO:187) (upper sequence) and the 80kDa polypeptide subunit of T. thermophila (SEQ ID NO:188) (lowersequence).

FIG. 37 shows a sequence comparison between the 123 kDa telomeraseprotein subunit of E. aediculatus (SEQ ID NO:189) (upper sequence) andthe 95 kDa telomerase polypeptide of T. thermophila (SEQ ID NO:190)(lower sequence).

FIG. 38 shows the best-fit alignment between a portion of the“La-domain” of the 43 kDa telomerase protein subunit of E. aediculatus(SEQ ID NO:191) (upper sequence) and a portion of the 95 kDa polypeptidesubunit of T. thermophila (SEQ ID NO:192) (lower sequence).

FIG. 39 shows the best-fit alignment between a portion of the“La-domain” of the 43 kDa telomerase protein subunit of E. aediculatus(SEQ ID NO:193) (upper sequence) and a portion of the 80 kDa polypeptidesubunit of T. thermophila (SEQ ID NO:194) (lower sequence).

FIG. 40 shows the alignment and motifs of the polymerase domain of the123 kDa telomerase protein subunit of E. aediculatus (SEQ ID NOS:38-41)and the polymerase domains of various reverse transcriptases including acytochrome oxidase group II intron 1-encoded protein from S. cerevisiaemitochondria (a1 S.c. (group II)) (SEQ ID NOS:204, 205, 54, 206, and56), Dong (LINE) (SEQ ID NOS:200-203), and yeast ESTp (L8543.12) (SEQ IDNOS:45, 46, 211 and 212), HIV-RT (SEQ ID NOS:207-210) and consensus (SEQID NOS:195-199).

FIG. 41 shows the alignment of a domain of the 43 kDa telomerase proteinsubunit (SEQ ID NO:213) with various La proteins (human La=SEQ IDNO:214; Xenopus LaA=SEQ ID NO:215; Drosophila La=SEQ ID NO:216; S.c.Lhplp=SEQ ID NO:217).

FIG. 42 shows the nucleotide sequence encoding the T. thermophila 80 kDaprotein subunit.

FIG. 43 shows the amino acid sequence of the T. thermophila 80 kDaprotein subunit (SEQ ID NO:219).

FIG. 44 shows the nucleotide sequence encoding the T. thermophila 95 kDaprotein subunit (SEQ ID NO:220).

FIG. 45 shows the amino acid sequence of the T. thermophila 95 kDaprotein subunit (SEQ ID NO:221).

FIG. 46 shows the amino acid sequence of L8543.12 (“Est2p”) (SEQ IDNO:222).

FIG. 47 shows the alignment of the amino acid sequence encoded by theOxytricha PCR product (SEQ ID NO:223) with the Euplotes p123 sequence(SEQ ID NO:224).

FIG. 48 shows the DNA sequence of Est2 (SEQ ID NO:225).

FIG. 49 shows partial amino acid sequence from a cDNA clone encodinghuman telomerase peptide motifs (SEQ ID NO:13).

FIG. 50 shows partial DNA sequence of a cDNA clone encoding humantelomerase peptide motifs (SEQ ID NO:8).

FIG. 51 shows the amino acid sequence of tez1, also called S. pombe trt(SEQ ID NO:112).

FIG. 52 shows, in two pages, the DNA sequence of tez1 (SEQ ID NO:111).Intronic and other non-coding regions are shown in lower case and exons(i.e., coding regions) are shown in upper case.

FIG. 53 shows the alignment of EST2p (SEQ ID NO:226), Euplotes (SEQ IDNO:227), and Tetrahymena SEQ ID NO:228) sequences, as well as consensussequence (SEQ ID NOS:229-231).

FIG. 54 shows the sequences of peptides (SEQ ID NOS:232-237) useful forproduction of anti-hTRT antibodies.

FIGS. 55A and B is a schematic summary of the tez1⁺ sequencingexperiments.

FIG. 56 shows two degenerate primers (SEQ ID NOS:238 and 241) used inPCR to identify the S. pombe homolog of the E. aediculatus p123sequences (SEQ ID NOS:239 and 240).

FIG. 57 shows the four major bands produced in PCR using degenerateprimers to identify the S. pombe homolog of the E. aediculatus p123sequences (SEQ ID NOS:239 and 240).

FIG. 58 shows the alignment of the M2 PCR product (SEQ ID NO:243) withE. aediculatus p123 (SEQ ID NO:242), S. cerevisiae (SEQ ID NO:244), andOxytricha (SEQ ID NO:223) telomerase protein sequences. Also shown arethe actual genomic sequences (SEQ ID NOS:246 and 249) and the peptidesencoded (SEQ ID NOS:245 and 250), degenerate primers Poly4 (SEQ IDNO:238) and Poly 1 (SEQ ID NO:244), and homologous regions of the M2 PCRproduct (SEQ ID NO:247) and its encoded peptide region (SEQ ID NO:248).

FIG. 59 is a schematic showing the 3′ RT PCR strategy for identifyingthe S. pombe homolog of the E. aediculatus p123.

FIGS. 60A, B, C and D shows characteristics of the libraries used toscreen for S. pombe telomerase protein sequences and shows the resultsof screening the libraries for S. pombe telomerase protein sequences.

FIG. 61 shows the positive results obtained with the HindIII-digestedpositive genomic clones containing S. pombe telomerase sequence.

FIG. 62 is a schematic showing the 5′ RT PCR strategy used to obtain afull length S. pombe TRT clone.

FIG. 63 shows the alignment of RT domains from telomerase catalyticsubunits for S. pombe (S.p.) (SEQ ID NOS:251-255), S. cerevisiae (S.c.)(SEQ ID NOS:256-260) and E. aediculatus (E.a.) (SEQ ID NOS:261-265).Consensus sequences=SEQ ID NOS:49 and 50.

FIG. 64 shows the alignment of the sequences from Euplotes (“Ea_p123”)(SEQ ID NO:110), S. cerevisiae (“Sc_Est2p”) (SEQ ID NO:222), and S.pombe (“SP_Tlp1p”) (SEQ ID NO:112). In Panel A, the shaded areasindicate residues shared between two sequences. In Panel B, the shadedareas indicate residues shared between all three sequences.

FIG. 65 shows the disruption strategy used with the telomerase genes inS. pombe.

FIG. 66 shows the experimental results confirming disruption of tez1.

FIG. 67 shows the progressive shortening of telomeres in S. pombe due totez1 disruption.

FIG. 68 shows, in four pages, the DNA (SEQ ID NO:266) and amino acid(SEQ ID NO:267) of the ORF encoding an approximately 63 kDa telomeraseprotein encoded by the EcoRI-NotI insert of clone 712562.

FIG. 69 shows an alignment of reverse transcriptase motifs from varioussources, E aediculatus p123 (SEQ ID NOS:268-273), S pombe tez1 (SEQ IDNOS:274-279), S. cerevisiae EST2 (SEQ ID NOS:280-285), and human Hs TCP1(SEQ ID NOS:286-291), with various consensus residues and motifsequences (SEQ ID NOS:49 and 50) indicated.

FIG. 70 provides a restriction and function map of plasmid pGRN121.

FIG. 71 shows, in two pages, the results of preliminary nucleic acidsequencing analysis of a hTRT cDNA sequence (SEQ ID NO:292).

FIG. 72 shows, in ten pages, the preliminary nucleic acid sequence ofhTRT (SEQ ID NO:292) and deduced ORF sequences in three reading frames(a=SEQ ID NOS:293-320; b=SEQ ID NOS:321-333; c=SEQ ID NOS:334-342).

FIG. 73 provides a restriction and function map of plasmid pGRN121.

FIG. 74 shows, in eight pages, refined nucleic acid sequence (SEQ IDNO:343) and deduced ORF sequences (SEQ ID NO:344) of hTRT

FIG. 75 shows a restriction map of lambda clone 25-1.1.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Telomerase is a ribonucleoprotein complex (RNP) comprising an RNAcomponent and a catalytic protein component. The present inventionrelates to the cloning and characterization of the catalytic proteincomponent of telomerase, hereinafter referred to as “TRT” (telomerasereverse transcriptase). TRT is so named because this protein acts as anRNA-dependent DNA polymerase (reverse transcriptase), using thetelomerase RNA component (hereinafter, “TR”) to direct synthesis oftelomere DNA repeat sequences. Moreover, TRT is evolutionarily relatedto other reverse transcriptases (see Example 12).

In one aspect, the present invention relates to the cloning andcharacterization of the catalytic protein component of human telomerase,hereinafter referred to as “hTRT.” Human TRT is of extraordinaryinterest and value because, as noted supra, telomerase activity in human(and other mammalian cells) correlates with cell proliferative capacity,cell immortality, and the development of a neoplastic phenotype. Forexample, telomerase activity, and, as demonstrated in Example 2, infra,levels of human TRT gene products and are elevated in immortal humancells (such as malignant tumor cells and immortal cell lines) relativeto mortal cells (such as most human somatic cells).

The present invention further provides methods and compositions valuablefor diagnosis, prognosis, and treatment of human diseases and diseaseconditions, as described in some detail infra. Also provided are methodsand reagents useful for immortalizing cells (in vivo and ex vivo),producing transgenic animals with desirable characteristics, andnumerous other uses, many of which are described infra. The inventionalso provides methods and reagents useful for preparing, cloning, orre-cloning TRT genes and proteins from ciliates, fungi, vertebrates,such as mammals, and other organisms.

As described in detail infra, TRT was initially characterized followingpurification of telomerase from the ciliate Euplotes aediculatus.Extensive purification of E. aediculatus telomerase, using RNA-affinitychromatography and other methods, yielded the protein Ap123″.Surprisingly, p123 is unrelated to proteins previously believed toconstitute the protein subunits of the telomerase holoenzyme (i.e., thep80 and p95 proteins of Tetrahymena thermophila). Analysis of the p123DNA and protein sequences (Genbank Accession No. U95964; FIGS. 13 and14) revealed reverse transcriptase (RT) motifs consistent with the roleof p123 as the catalytic subunit of telomerase (see, e.g., FIGS. 1, 4and 11). Moreover, p123 is related to a S. cerevisiae (yeast) protein,Est2p, which was known to play a role in maintenance of telomeres in S.cerevisiae (Genbank Accession No. S5396), but prior to the presentinvention was not recognized as encoding a telomerase catalytic subunitprotein (see, e.g., Lendvay et al., 1996, Genetics, 144:1399).

In one aspect, the present invention provides reagents and methods foridentifying and cloning novel TRTs using: nucleic acid probes andprimers generated or derived from the TRT polynucleotides disclosed(e.g., for cloning TRT genes and cDNAs); antibodies that specificallyrecognize the motifs or motif sequences or other TRT epitopes (e.g., forexpression cloning TRT genes or purification of TRT proteins); byscreening computer databases; or other means. For example, as describedin Example 1, PCR (polymerase chain reaction) amplification of S. pombeDNA was carried out with degenerate-sequence primers designed from theEuplotes p123 RT motifs B′ and C. Of four prominent products generated,one encoded a peptide sequence homologous to Euplotes p123 and S.cerevisiae Est2p. Using this PCR product as a probe, the completesequence of the S. pombe TRT homologue was obtained by screening of S.pombe cDNA and genomic libraries and amplifying S. pombe RNA by reversetranscription and PCR (RT-PCR). The complete sequence of the S. pombegene (“trt1”; GenBank Accession No. AF015783; FIG. 15) revealed thathomology with p123 and Est2p was especially high in the reversetranscriptase motifs. S. pombe trt1 is also referred to as tez1.

Amplification using degenerate primers derived from the telomerase RTmotifs was also used to obtain TRT gene sequences in Oxytricha trifallaxand Tetrahymena thermophila, as described in Example 1.

The Euplotes p123, S. pombe trt1, and S. cerevisiae Est2p nucleic acidsequences of the invention were used in a search of a computerizeddatabase of human expressed sequence tags (ESTs) using the program BLAST(Altschul et al. al, 1990, J. Mol. Biol. 215:403). Searching thisdatabase with the Est2p sequence did not indicate a match, but searchingwith p123 and trt1 sequences identified a human EST (Genbank accessionno. AA281296; see SEQ ID NO:8), as described in Example 1, putativelyencoding a homologous protein. Complete sequencing of the cDNA clonecontaining the EST (hereinafter, “clone 712562”; see SEQ ID NO:3) showedthat seven RT motifs were present. However, this clone did not encode acontiguous human TRT with all seven motifs, because motifs B′, C, D, andE were contained in a different open reading frame (ORF) than the moreNH₂-terminal motifs. In addition, the distance between motifs A and B′was substantially shorter than that of the three previouslycharacterized TRTs. Clone 712562 was obtained from the I.M.A.G.E.Consortium; Lennon et al., 1996, Genomics 33:151.

A cDNA clone, pGRN121, encoding a functional hTRT (see FIG. 16, SEQ IDNO:1) was isolated from a cDNA library derived from the human 293 cellline as described in Example 1. Comparing clone 712562 with pGRN121showed that clone 712562 has a 182 base pair (see FIG. 24, SEQ ID NO:9)deletion between motifs A and B′. The additional 182 base pairs presentin pGRN121 place all of the TRT motifs in a single open reading frame,and increase the spacing between the motif A and motif B′ regions to adistance consistent with the other known TRTs. As is described infra inthe Examples (e.g., Example 7), SEQ ID NO:1 encodes a catalyticallyactive telomerase protein having the sequence of SEQ ID NO:2. Thepolypeptide of SEQ ID NO:2 has 1132 residues and a calculated molecularweight of about 127 kilodaltons (kD).

As is discussed infra, and described in Example 9, infra, TRT cDNAspossessing the 182 basepair deletion characteristic of the clone 712562are detected following reverse transcription of RNA fromtelomerase-positive cells (e.g., testis and 293 cells). hTRT RNAslacking this 182 base pair sequence are referred to generally as “Δ182variants” and may represent one, two, or several species. Although thehTRT variants lacking the 182 basepair sequence found in the pGRN121cDNA are unlikely to encode a fully active telomerase catalytic enzyme,they may play a role in telomerase regulation, as discussed infra,and/or have partial telomerase activity, such as telomere binding or hTRbinding activity, as discussed infra.

Thus, in one aspect, the present invention provides an isolatedpolynucleotide with a sequence of a naturally occurring human TRT geneor mRNA including, but not limited to, a polynucleotide having thesequence as set forth in FIG. 16 (SEQ ID NO:1). In a related aspect, theinvention provides a polynucleotide encoding an hTRT protein, fragment,variant or derivative. In another related aspect, the invention providessense and antisense nucleic acids that bind to an hTRT gene or mRNA. Theinvention further provides hTRT proteins, whether synthesized orpurified from natural sources, as well as antibodies and other agentsthat specifically bind an hTRT protein or a fragment thereof. Thepresent invention also provides many novel methods, including methodsthat employ the aforementioned compositions, for example, by providingdiagnostic and prognostic assays for human diseases, methods fordeveloping therapeutics and methods of therapy, identification oftelomerase-associated proteins, and methods for screening for agentscapable of activating or inhibiting telomerase activity. Numerous otheraspects and embodiments of the invention are provided infra.

One aspect of the invention is the use of a polynucleotide that is atleast ten nucleotides to about 10 kb or more in length and comprises acontiguous sequence of at least ten nucleotides that is identical orexactly complementary to a contiguous sequence in a naturally occurringhTRT gene or hTRT mRNA in assaying or screening for an hTRT genesequence or hTRT mRNA, or in preparing a recombinant host cell.

A further aspect of the invention is the use of an agent increasingexpression of hTRT in the manufacture of a medicament for the treatmentof a condition addressed by increasing proliferative capacity of avertebrate cell, optionally the medicament being for inhibiting theeffects of aging.

Yet a further aspect of the invention is the use of an inhibitor oftelomerase activity in the manufacture of a medicament for the treatmentof a condition associated with an elevated level of telomerase activitywithin a human cell.

The proteins, variants and fragments of the invention, and the encodingpolynucleotides or fragments, are also each provided in a further aspectof this invention for use as a pharmaceutical.

The invention further includes the use of a protein, variant orfragment, or of a polynucleotide or fragment, in each case as definedherein, in the manufacture of a medicament, for example in themanufacture of a medicament for inhibiting an effect of aging or cancer.

Another aspect of the invention is a polynucleotide selected from:

(a) the DNA having a sequence as set forth in FIG. 16;

(b) a polynucleotide of at least 10 nucleotides which hybridizes to theforegoing DNA and which codes for an hTRT protein or variant or whichhybridizes to a coding sequence for such a variant; and,

(c) DNA sequences which are degenerate as a result of the genetic codeto the DNA sequences defined in (a) and (b) and which code for an hTRTpolypeptide or variant.

In certain embodiments of the present invention, the hTRTpolynucleotides are other than the 389 nucleotide polynucleotide of SEQID NO:8 and/or other than clone 712562, the plasmid containing aninsert, the sequence of which insert is shown in FIG. 18 (SEQ ID NO:3).

The description below is organized by topic. Part II further describesamino acid motifs characteristic of TRT proteins, as well as TRT genesencoding proteins having such motifs. Parts III-VI describe, inter alia,nucleic acids, proteins, antibodies and purified compositions of theinvention with particular focus on human TRT related compositions. PartVII describes, inter alia, methods and compositions of the inventionuseful for treatment of human disease. Part VIII describes productionand identification of immortalized human cell lines. Part IX describes,inter alia, uses of the nucleic acids, polynucleotides, and othercompositions of the invention for diagnosis of human diseases. Part Xdescribes, inter alia, methods and compositions of the invention usefulfor screening and identifying agents and treatments that modulate (e.g.,inhibit or promote) telomerase activity or expression. Part XIdescribes, inter alia, transgenic animals (e.g., telomerase knockoutanimals and cells). Part XII is a glossary of terms used in Parts I-XI.Part XIII describes examples relating to specific embodiments of theinvention. The organization of the description of the invention by topicand subtopic is to provide clarity, and not to be limiting in any way.

II. TRT Genes and Proteins

The present invention provides isolated and/or recombinant genes andproteins having a sequence of a telomerase catalytic subunit protein(i.e., telomerase reverse transcriptase), including, but not limited to,the naturally occurring forms of such genes and proteins in isolated orrecombinant form. Typically, TRTs are large, basic, proteins havingreverse transcriptase (RT) and telomerase-specific (T) amino acidmotifs, as disclosed herein. Because these motifs are conserved acrossdiverse organisms, TRT genes of numerous organisms may be obtained usingthe methods of the invention or identified using primers, nucleic acidprobes, and antibodies of the invention, such as those specific for oneor more of the motif sequences.

The seven RT motifs found in TRTs, while similar to those found in otherreverse transcriptases, have particular hallmarks. For example, as shownin FIG. 4, within the TRT RT motifs there are a number of amino acidsubstitutions (marked with arrows) in residues highly conserved amongthe other RTs. For example, in motif C the two aspartic acid residues(DD) that coordinate active site metal ions (see, Kohlstaedt et al.,1992, Science 256:1783; Jacobo-Molina et al., 1993, Proc. Natl. AcadSci. U.S.A. 90:6320; Patel et al., 1995, Biochemistry 34:5351) occur inthe context hxDD(F/Y) (SEQ ID NO:345) in the telomerase RTs compared to(F/Y)xDDh (SEQ ID NO:346) in the other RTs (where “h” is a hydrophobicamino acid, and “x” is any amino acid; see Xiong et al., 1990, EMBO J.9:3353; Eickbush, in The Evolutionary Biology of Viruses, (S. Morse,Ed., Raven Press, NY, p. 121, 1994)). Another systematic changecharacteristic of the telomerase subgroup occurs in motif E, whereWxGxSx (SEQ ID NO:347) is a consensus sequence or is conserved among thetelomerase proteins, whereas hLGxxh (SEQ ID NO:348) is characteristic ofother RTs (Xiong et al., supra; Eickbush supra). This motif E is calledthe “primer grip”, and mutations in this region have been reported toaffect RNA priming but not DNA priming (Powell et al., 1997, J. Biol.Chem. 272:13262). Because telomerase requires a DNA primer (e.g., thechromosome 3′ end), it is not unexpected that telomerase should differfrom other RTs in the primer grip region. In addition, the distancebetween motifs A and B′ is longer in the TRTs than is typical for otherRTs, which may represent an insertion within the “fingers” region of thestructure which resembles a right hand (FIG. 3; see Kohlstaedt et al.,supra; Jacobo-Molina et al., supra; and Patel et al., supra).

Moreover, as noted supra, Motif T is an additional hallmark of TRTproteins. This Motif T, as shown, for example in FIG. 4(W-L-X-Y-X-X-h-h-X-h-h-X-p-F-F-Y-X-T-E-X-p-X-X-X-p-X-X-X-Y-X-R-K-X-X-W(SEQ ID NO:349) [X is any amino acid, h is hydrophobic, p is polar]),comprises a sequence that can be described using the formula:

Trp-R₁-X₇-R₁-R₁-R₂-X-Phe-Phe-Tyr-X-Thr-Glu-X₈₋₉-R₃-R₃-Arg-R₄-X₂-Trp (SEQID NOS:11 and 12)

where X is any amino acid and the subscript refers to the number ofconsecutive residues, R₁ is leucine or isoleucine, R₂ is glutamine orarginine, R₃ is phenylalanine or tyrosine, and R₄ is lysine orhistidine.

The T motif can also be described using the formula:

Trp-R₁-X₄-h-h-X-h-h-R₂-p-Phe-Phe-Tyr-X-Thr-Glu-X-p-X₃-p-X₂₋₃-R₃-R₃-Arg-R₄-X₂-Trp(SEQ ID NOS:350 and 351)

where X is any amino acid and a subscript refers to the number ofconsecutive residues, R₁ is leucine or isoleucine, R₂ is glutamine orarginine, R₃ is phenylalanine or tyrosine, R₄ is lysine or histidine, his a hydrophobic amino acid selected from Ala, Leu, lie, Val, Pro, Phe,Trp, and Met, and p is a polar amino acid selected from Gly, Ser, Thr,Tyr, Cys, Asn and Gln.

In one embodiment, the present invention provides isolated naturallyoccurring and recombinant TRT proteins comprising one or more of themotifs illustrated in FIG. 11, e.g.,

Motif T W-X₁₂-FFY-X-TE-X₁₀₋₁₁-R-X₃-W-X₇-I (SEQ ID NOS:352 and 353) MotifT′ E-X₂-V-X (SEQ ID NO:354) Motif 1 X₃-R-X₂-P-K-X₃, (SEQ D NO:355) or,alternatively, h-R-h-X-P-K (SEQ ID NO:633) Motif 2 X-R-X-I-X (SEQ IDNO:356) or, alternatively, (F/L)-R-h-I-X₂-h (SEQ ID NO:634) Motif AX4-F-X₃-D-X4-YD-X₂, (SEQ ID NO:357) or, alternatively,P-X-L-Y-F-h-X-h-D-h-X₂-C-Y-D-X-I (SEQ ID NO:635) Motif B′Y-X₄-G-X₂-QG-X₃-S-X₈ (SEQ ID NO:358) or, alternatively,K-X-Y-X-Q-X₂-G-I-P-Q-G-S-X-L-S-X-h-L (SEQ ID NO:636) Motif CX₆-DD-X-L-X₃, (SEQ ID NO:359) or, altematively, L-L-R-L-X-D-D-X-L-h-I-T(SEQ ID NO:637)

When the TRT protein shown contains more than one TRT motif, the order(NH2 ->COOH) is as shown in FIG. 11.

In one embodiment, the present invention provides isolated naturallyoccurring TRT proteins comprising the following supermotif:(NH₂)-X₃₀₀₋₆₀₀-W-X₁₂-FFY-X-TE-X₁₀₋₁₁-R-X₃-W-X₇-I-X₅₋₂₀-E-X₂-V-X-X₅₋₂₀-X₃-R-X₂-PK-X₄₋₁₀-R-X-I-X-X₆₀₋₈₀-X₄-F-X₃-D-X₄-YD-X₂-X₈₀₋₁₃₀-Y-X₄-G-X₂-QG-X₃-S-X₈-X₅₋₃₅-X₆-DD-X-L-X₃-X₁₀₋₂₀-X₁₂-K(SEQ ID NO:727)

It will be apparent to one of skill that, provided with the reagents,including the TRT sequences disclosed herein for those reagents and themethods and guidance provided herein (including specific methodologiesdescribed infra), TRT genes and proteins can be obtained, isolated andproduced in recombinant form by one of ordinary skill. For example,primers (e.g., degenerate amplification primers) are provided thathybridize to gene sequences encoding RT and T motifs characteristic ofTRT. For example, one or more primers or degenerate primers thathybridize to sequences encoding the FFYXTE (SEQ ID NO:360) region of theT motif, other TRT motifs (as discussed infra), or combinations ofmotifs or consensus sequences, can be prepared based on the codon usageof the target organism, and used to amplify the TRT gene sequence fromgenomic DNA or cDNA prepared from the target organism. Use of degenerateprimers is well known in the art and entails use of sets of primers thathybridize to the set of nucleic acid sequences that can potentiallyencode the amino acids of the target motif, taking into account codonpreferences and usage of the target organism, and by using amplification(e.g., PCR) conditions appropriate for allowing base mismatches in theannealing steps of PCR. Typically two primer sets are used; however,single primer (or, in this case, a single degenerate primer set)amplification systems are well known and may be used to obtain TRTgenes.

Table 1 provides illustrative primers of the invention that may be usedto amplify novel TRT nucleic acids, particularly those from vertebrates(e.g., humans and other mammals). “N” is an equimolar mixture of allfour nucleotides, and nucleotides within parentheses are equimolarmixtures of the specified nucleotides.

TABLE 1 ILLUSTRATIVE DEGENERATE PRIMERS FOR AMPLIFICATION OF TRT NUCLEICACIDS motif primer motif SEQ ID NO: direction 5′ sequence-3′ SEQ ID NO:a FFYVTE 361 Forward TT(CT)TT(CT)TA(CT)GTNACNGA 362 b FFYVTE 361 ReverseTCNGTNAC(GA)TA(GA)AA(GA)AA 363 c RFIPKP 364 Forward(CA)GNTT(CT)AT(ACT)CCNAA(AG)CC 365 d RFIPKP 364 ReverseGG(TC)TTNGG(TGA)AT(GA)AANC 366 e AYDTI 367 Forward GCNTA(CT)GA(CT)ACNAT365 f AYDTI 367 Reverse TANGT(GA)TC(GA)TANGC 369 g GIPQG 370 ForwardGGNAT(ACT)CCNCA(AG)GG 371 h GIPQGS 21 Reverse(GC)(AT)NCC(TC)TGNGG(TGA)ATNCC 372 i LVDDFL 373 Forward(CT)TNGTNGA(CT)GA(CT)TT(CT)(CT)T 374 j DDFLLVT 375 ReverseGTNACNA(GA)NA(GA)(GA)AA(GA)TC(GA)TC 376 Preferred primer combinations (y= yes, n = no) Reverse Forward b d f h i a- n y y y y c- n n y y y e- nn n y y g- n n n n y i- n n n n n

In one embodiment, an amplified TRT nucleic acid is used as ahybridization probe for colony hybridization to a library (e.g., cDNAlibrary) made from the target organism, such that a nucleic acid havingthe entire TRT protein coding sequence, or a substantial portionthereof, is identified and isolated or cloned. Reagents and methods suchas those just described were used in accordance with the methodsdescribed herein to obtain TRT gene sequences of Oxytricha trifallax andTetrahymena thermophila, as described in detail infra. It will berecognized that following cloning of a previously uncharacterized TRTgene, the sequence can be determined by routine methods and the encodedpolypeptide synthesized and assayed for a TRT activity, such astelomerase catalytic activity (as described herein and/or by telomeraseassays known in the art).

It will also be apparent to those of skill that TRT genes may be clonedusing any of a variety of cloning methods of the invention because theTRT motif sequences and the nucleic acids of the invention comprisingsuch sequences can be used in a wide variety of such methods. Forexample, hybridization using a probe based on the sequence of a knownTRT to DNA or other nucleic acid libraries from the target organism, asdescribed in Example 1 can be used. It will be appreciated thatdegenerate PCR primers or their amplification products such as thosedescribed supra, may themselves be labeled and used as hybridizationprobes. In another embodiment, expression cloning methods are used. Forexample, one or more antibodies that specifically bind peptides thatspan a TRT motif or other TRT epitope, such as the FFYXTE (SEQ IDNO:360) motif can be employed to isolate a ribosomal complex comprisinga TRT protein and the mRNA that encodes it. For generating suchantibodies of the invention, the peptide immunogens are typicallybetween 6 and 30 amino acids in length, more often about 10 to 20 aminoacids in length. The antibodies may also be used to probe a cDNAexpression library derived from the organism of interest to identify aclone encoding a TRT sequence. In another embodiment, computer searchesof DNA databases for DNAs containing sequences conserved with known TRTscan also be used to identify a clone comprising TRT sequence.

In one aspect, the present invention provides compositions comprising anisolated or recombinant polypeptide having the amino acid sequence of anaturally occurring TRT protein. Usually the naturally occurring TRT hasa molecular weight of between about 80,000 daltons (D) and about 150,000D, most often between about 95,000 D and about 130,000 D. Typically, thenaturally occurring TRT has a net positive charge at pH 7 (calculated pItypically greater than 9). In one embodiment, the polypeptide exhibits atelomerase activity as defined herein. In a related embodiment, thepolypeptide has a TRT-specific region (T motif) sequence and exhibits atelomerase activity. The invention further provides fragments of suchpolypeptides. The present invention also provides isolated orrecombinant polynucleotide having the sequence of a naturally occurringgene encoding a TRT protein. The invention provides regents useful forisolating sequence of a TRT from nonvertebrate (such as a yeast) andvertebrates, such as mammals (e.g., murine or human). The isolatedpolynucleotide may be associated with other naturally occurring orrecombinant or synthetic vector nucleic acid sequences. Typically, theisolated nucleic acid is smaller than about 300 kb, often less thanabout 50 kb, more often less than about 20 kb, frequently less thanabout 10 kb and sometimes less than about 5 kb or 2 kb in length. Insome embodiments the isolated TRT polynucleotide is even smaller, suchas a gene fragment, primer, or probe of less than about 1 kb or lessthan 0.1 kb.

III. Nucleic Acids

A) Generally

The present invention provides isolated and recombinant nucleic acidshaving a sequence of a polynucleotide encoding a telomerase catalyticsubunit protein (TRT), such as a recombinant TRT gene from Euplotes,Tetrahymena, S. pombe or humans. Exemplary polynucleotides are providedin FIG. 13 (Euplotes); FIG. 15 (S. pombe) and FIG. 16 (human, GenBankAccession No. AF015950). The present invention provides sense andanti-sense polynucleotides having a TRT gene sequence, including probes,primers, TRT-protein-encoding polynucleotides, and the like.

B) Human TRT

The present invention provides nucleic acids having a sequence of atelomerase catalytic subunit from humans (i.e., hTRT).

In one aspect, the invention provides a polynucleotide having a sequenceor subsequence of a human TRT gene or RNA. In one embodiment, thepolynucleotide of the invention has a sequence of SEQ ID NO:1 shown inFIG. 16 or a subsequence thereof. In another embodiment, thepolynucleotide has a sequence of SEQ ID NO:3 (FIG. 18), SEQ ID NO:4(FIG. 20), or subsequences thereof. The invention also providespolynucleotides with substantial sequence identity to the hTRT nucleicacid sequences disclosed herein, e.g., including but not limited to SEQID NOS:1 [FIG. 16], 4 [FIG. 20], 6 [FIG. 21], and 7 [FIG. 12]). Thus,the invention provides naturally occurring alleles of human TRT genesand variant polynucleotide sequences having one or more nucleotidedeletions, insertions or substitutions relative to an hTRT nucleic acidsequence disclosed herein. As described infra, variant nucleic acids maybe produced using the recombinant or synthetic methods described belowor by other means.

The invention also provides isolated and recombinant polynucleotideshaving a sequence from a flanking region of a human TRT gene. Suchpolynucleotides include those derived from genomic sequences ofuntranslated regions of the hTRT mRNA. An exemplary genomic sequence isshown in FIG. 21 (SEQ ID NO:6). As described in Example 4, SEQ ID NO:6was obtained by sequencing a clone, λGΦ5 isolated from a human genomiclibrary. Lambda GΦ5 contains a 15 kilobasepair (kbp) insert includingapproximately 13,000 bases 5′ to the hTRT coding sequences. This clonecontains hTRT promoter sequences and other hTRT gene regulatorysequences (e.g., enhancers).

The invention also provides isolated and recombinant polynucleotideshaving a sequence from an intronic region of a human TRT gene. Anexemplary intronic sequence is shown in FIG. 12 (SEQ ID NO:7; seeExample 3). In some embodiments, hTRT introns are included in“minigenes” for improved expression of hTRT proteins in eukaryoticcells.

In a related aspect, the present invention provides polynucleotides thatencode hTRT proteins or protein fragments, including modified, alteredand variant hTRT polypeptides. In one embodiment, the encoded hTRTprotein or fragment has an amino acid sequence as set forth in FIG. 17(SEQ ID NO:2), or with conservative substitutions of SEQ ID NO:2. In oneembodiment, the encoded hTRT protein or fragment has substitutions thatchange an activity of the protein (e.g., telomerase catalytic activity).

It will be appreciated that, as a result of the degeneracy of thegenetic code, the nucleic acid encoding the hTRT protein need not havethe sequence of a naturally occurring hTRT gene, but that a multitude ofpolynucleotides can encode an hTRT polypeptide having an amino acidsequence of SEQ ID NO:2. The present invention provides each and everypossible variation of nucleotide sequence that could be made byselecting combinations based on possible codon choices made inaccordance with known triplet genetic codes, and all such variations arespecifically disclosed hereby. Thus, although in some cases hTRTpolypeptide-encoding nucleotide sequences that are capable ofhybridizing to the nucleotide sequence of the naturally occurringsequence (under appropriately selected conditions of stringency) arepreferred, it may be advantageous in other cases to produce nucleotidesequences encoding hTRT that employ a substantially different codonusage and so perhaps do not hybridize to nucleic acids with thenaturally occurring sequence.

In particular embodiments, the invention provides hTRT oligo- andpolynucleotides that comprise a subsequence of an hTRT nucleic aciddisclosed herein (e.g., SEQ ID NOS:1 and 6). The nucleic acids of theinvention typically comprise at least about 10, more often at leastabout 12 or about 15 consecutive bases of the exemplified hTRTpolynucleotide. Often, the nucleic acid of the invention will comprise alonger sequence, such as at least about 25, about 50, about 100, about200, or at least about 500 to 3000 bases in length, for example whenexpression of a polypeptide, or full length hTRT protein is intended.

In still other embodiments, the present invention provides “Δ182 Htrt”polynucleotides having a sequence identical or complementary tonaturally occurring or non-naturally occurring hTRT polynucleotides suchas SEQ ID NO:3 or SEQ ID NO:4, which do not contain the 182 nucleotidesequence (SEQ ID NO:9) found in pGRN121 (and also absent in clone712562). These polynucleotides are of interest, in part, because theyencode polypeptides that contain different combinations or arrangementsof TRT motifs than found in the “full-length” hTRT polypeptide (SEQ IDNO:2) such as is encoded by pGRN121. As discussed infra, it iscontemplated that these polypeptides may play a biological role innature (e.g., in regulation of telomerase expression in cells) and/orfind use as therapeutics (e.g., as dominant-negative products thatinhibit function of wild-type proteins), or have other roles and uses,e.g. as described herein.

For example, in contrast to the polypeptide encoded by pGRN121, clone712562 encodes a 259 residue protein with a calculated molecular weightof approximately 30 kD (hereinafter, “712562 hTRT”). The 712562 hTRTpolypeptide (SEQ ID NO:10 [FIG. 19]) contains motifs T, 1, 2, and A, butnot motifs B′, C, D and E (See FIG. 4). Similarly, a variant hTRTpolypeptide with therapeutic and other activities may be expressed froma nucleic acid similar to the pGRN121 cDNA but lacking the 182 basepairsmissing in clone 712562, e.g., having the sequence shown in FIG. 20 (SEQID NO:4). This nucleic acid (hereinafter, “pro90 hTRT”), which may besynthesized using routine synthetic or recombinant methods as describedherein, encodes a protein of 807 residues (calculated molecular weightof approximately 90 kD) that shares the same amino terminal sequence asthe hTRT protein encoded by SEQ ID NO:1, but diverges at thecarboxy-terminal region (the first 763 residues are common, the last 44residues of pro90 hTRT are different than “full-length” hTRT). The pro90hTRT polypeptide contains motifs T, 1, 2, and A, but not motifs B, C, D,E, and thus may have some, but not likely all telomerase activities.

C) Production of Human TRT Nucleic Acids

The polynucleotides of the invention have numerous uses including, butnot limited to, expression of polypeptides encoding hTRT or fragmentsthereof, use as sense or antisense probes or primers for hybridizationand/or amplification of naturally occurring hTRT genes or RNAs (e.g. fordiagnostic or prognostic applications), and as therapeutic agents (e.g.,in antisense, triplex, or ribozyme compositions). As will be apparentupon review of the disclosure, these uses will have enormous impact onthe diagnosis and treatment of human diseases relating to aging, cancer,and fertility as well as the growth, reproduction, and manufacture ofcell-based products. As described in the following sections, the hTRTnucleic acids of the invention may be made (e.g., cloned, synthesized,or amplified) using techniques well known in the art.

1) Cloning, Amplification, and Recombinant Production

In one embodiment, hTRT genes or cDNAs are cloned using a nucleic acidprobe that specifically hybridizes to an hTRT mRNA, cDNA, or genomicDNA. One suitable probe for this purpose is a polynucleotide having allor part of the sequence provided in FIG. 16 (SEQ ID NO:1), such as aprobe comprising a subsequence thereof. Typically, the target hTRTgenomic DNA or cDNA is ligated into a vector (e.g., a plasmid, phage,virus, yeast artificial chromosome, or the like) and may be isolatedfrom a genomic or cDNA library (e.g., a human placental cDNA library).Once an hTRT nucleic acid is identified, it can be isolated according tostandard methods known to those of skill in the art. An illustrativeexample of screening a human cDNA library for the hTRT gene is providedin Example 1; similarly, an example of screening a human genomic libraryis found in Examples 3 and 4. Cloning methods are well known and aredescribed, for example, in Sambrook et al., (1989) Molecular Cloning: ALaboratory Manual, 2nd ed., Vols. 1-3, Cold Spring Harbor Laboratoryhereinafter, “Sambrook”); Berger and Kimmel, (1987) Methods inEnzymology, Vol. 152: Guide to Molecular Cloning Techniques, San Diego:Academic Press, Inc.; Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing and Wiley-Interscience, New York (1997);Cashion et al., U.S. Pat. No. 5,017,478; and Carr, European Patent No.0,246,864.

The invention also provides hTRT genomic or cDNA nucleic acids isolatedby amplification methods such as the polymerase chain reaction (PCR). Inone embodiment, hTRT protein coding sequence is amplified from an RNA orcDNA sample (e.g., double stranded placental cDNA (Clontech, Palo AltoCalif.)) using the primers 5′-GTGAAGGCACTGTTCAGCG-3′ (“TCP1.1”) (SEQ IDNO:377) and 5′-CGCGTGGGTGAGGTGAGGTG-3 (“TCP 1.15”) (SEQ ID NO:378). Insome embodiments a third primer or second pair of primers may be used,e.g., for “nested PCR”, to increase specificity. One example of a secondpair of primers is 5′-CTGTGCTGGGCCTGGACGATA-3′ (“TCP1.14”) (SEQ IDNO:379) and 5′-AGCTTGTTCTCCATGTCGCCGTAG-3′ (“billTCP6”) (SEQ ID NO:380).It will be apparent to those of skill that numerous other primers andprimer combinations, useful for amplification of hTRT nucleic acids areprovided by the present invention.

Moreover, the invention provides primers that amplify any specificregion (e.g., coding regions, promoter regions, and/or introns) orsubsequence of hTRT genomic DNA, cDNA or RNA. For example, the hTRTintron at position 274/275 of SEQ ID NO:1 (see Example 3) may beamplified (e.g., for detection of genomic clones) using primers TCP1.57and TCP1.52 (primer pair 1) or primers TCP1.49 and TCP1.50 (primer pair2). (Primer names refer to primers listed in Table 2, infra.) The primerpairs can be used individually or in a nested PCR where primer set 1 isused first. Another illustrative example relates to primers thatspecifically amplify and so detect the 5′ end of the hTRT mRNA or theexon encoding the 5′ end of hTRT gene (e.g., to assess the size orcompleteness of a cDNA clone). The following primer pairs are useful foramplifying the 5′ end of hTRT: primers K320 and K321 (primer pair 3);primers K320 and TCP1.61 (primer pair 4); primers K320 and K322 (primerpair 5). The primer sets can be used in a nested PCR in the order set 5,then set 4 or set 3, or set 4 or set 5, then set 3. Yet anotherillustrative example involves primers chosen to amplify or detectspecifically the conserved hTRT TRT motif region comprisingapproximately the middle third of the mRNA (e.g., for use as ahybridization probe to identify TRT clones from, for example, nonhumanorganisms). The following primer pairs are useful for amplifying the TRTmotif region of hTRT nucleic acids: primers K304 and TCP1.8 (primer pair6), or primers Lt1 and TCP1.15 (primer pair 7). The primer sets can beused in a nested PCR experiment in the order set 6 then set 7.

Suitable PCR amplification conditions are known to those of skill andinclude (but are not limited to) 1 unit Taq polymerase (Perkin Elmer,Norwalk Conn.), 100 μM each dNTP (dATP, dCTP, dGTP, dTTP), 1×PCR buffer(50 mM KCl, 10 mM Tris, pH 8.3 at room temperature, 1.5 mM MgCl₂, 0.01%gelatin) and 0.5 μM primers, with the amplification run for about 30cycles at 94° for 45 sec, 55° for 45 sec and 72° for 90 sec. It will berecognized by those of skill in the art that other thermostable DNApolymerases, reaction conditions, and cycling parameters will alsoprovide suitable amplification. Other suitable in vitro amplificationmethods that can be used to obtain hTRT nucleic acids include, but arenot limited to, those herein, infra. Once amplified, the hTRT nucleicacids can be cloned, if desired, into any of a variety of vectors usingroutine molecular biological methods or detected or otherwise utilizedin accordance with the methods of the invention.

One of skill will appreciate that the cloned or amplified hTRT nucleicacids obtained as described above can be prepared or propagated usingother methods, such as chemical synthesis or replication bytransformation into bacterial systems, such as E. coli (see, e.g.,Ausubel et al., supra), or eukaryotic, such as mammalian, expressionsystems. Similarly, hTRT RNA can be expressed in accordance with thepresent in vitro methods, or in bacterial systems such as E. coli using,for example, commercially available vectors containing promotersrecognized by an RNA polymerase such as T7, T3 or SP6, or transcriptionof DNA generated by PCR amplification using primers containing an RNApolymerase promoter.

The present invention further provides altered or modified hTRT nucleicacids. It will be recognized by one of skill that the cloned oramplified hTRT nucleic acids obtained can be modified (e.g., truncated,derivatized, altered) by methods well known in the art (e.g.,site-directed mutagenesis, linker scanning mutagenesis) or simplysynthesized de novo as described below. The altered or modified hTRTnucleic acids are useful for a variety of applications, including, butnot limited to, facilitating cloning or manipulation of an hTRT gene orgene product, or expressing a variant hTRT gene product. For example, inone embodiment, the hTRT gene sequence is altered such that it encodesan hTRT polypeptide with altered properties or activities, as discussedin detail in infra, for example, by mutation in a conserved motif ofhTRT. In another illustrative example, the mutations in the proteincoding region of an hTRT nucleic acid may be introduced to alterglycosylation patterns, to change codon preference, to produce splicevariants, remove protease-sensitive sites, create antigenic domains,modify specific activity, and the like. In other embodiments, thenucleotide sequence encoding hTRT and its derivatives is changed withoutaltering the encoded amino acid sequences, for example, the productionof RNA transcripts having more desirable properties, such as increasedtranslation efficiency or a greater or a shorter half-life, compared totranscripts produced from the naturally occurring sequence. In yetanother embodiment, altered codons are selected to increase the rate atwhich expression of the peptide occurs in a particular prokaryotic oreukaryotic expression host in accordance with the frequency with whichparticular codons are utilized by the host. Useful in vitro and in vivorecombinant techniques that can be used to prepare variant hTRTpolynucleotides of the invention are found in Sambrook et al. andAusubel et al., both supra.

As noted supra, the present invention provides nucleic acids havingflanking (5′ or 3′) and intronic sequences of the hTRT gene. The nucleicacids are of interest, inter alia, because they contain promoter andother regulatory elements involved in hTRT regulation and useful forexpression of hTRT and other recombinant proteins or RNA gene products.It will be apparent that, in addition to the nucleic acid sequencesprovided in SEQ ID NOS:6 and 7, additional hTRT intron and flankingsequences may be readily obtained using routine molecular biologicaltechniques. For example, additional hTRT genomic sequence may beobtained from Lambda clone GΦ5 (ATCC Accession No. 209024), describedsupra and in Example 4. Still other hTRT genomic clones and sequencesmay be obtained by screening a human genomic library using an hTRTnucleic acid probe having a sequence or subsequence from SEQ ID NO:1.Additional clones and sequences (e.g., still further upstream) may beobtained by using labeled sequences or subclones derived from λGΦ5 toprobe appropriate libraries. Other useful methods for furthercharacterization of hTRT flanking sequences include those generalmethods described by Gobinda et al., 1993, PCR Meth. Applic. 2:318;Triglia et al., 1988, Nucleic Acids Res. 16:8186; Lagerstrom et al.,1991, PCR Methods Applic. 1:111; and Parker et al., 1991, Nucleic AcidsRes. 19:3055.

Intronic sequences can be identified by routine means such as bycomparing the hTRT genomic sequence with hTRT cDNA sequences (see, e.g.,Example 3), by S1 analysis (see Ausubel et al., supra, at Chapter 4), orvarious other means known in the art. Intronic sequences can also befound in pre-mRNA (i.e., unspliced or incompletely spliced mRNAprecursors), which may be amplified or cloned following reversetranscription of cellular RNA.

When desired, the sequence of the cloned, amplified, or otherwisesynthesized hTRT or other TRT nucleic acid can be determined or verifiedusing DNA sequencing methods well known in the art (see, e.g., Ausubelet al., supra). Useful methods of sequencing employ such enzymes as theKlenow fragment of DNA polymerase I, Sequenase (US Biochemical Corp,Cleveland Ohio), Taq DNA polymerase (Perkin Elmer, Norwalk Conn.),thermostable T7 polymerase (Amersham, Chicago Ill.), or combinations ofrecombinant polymerases and proofreading exonucleases such as theELONGASE Amplification System marketed by Gibco BRL (Gaithersburg Md.).When sequencing or verifying the sequence of oligonucleotides (such asoligonucleotide made de novo by chemical synthesis), the method of Maxamand Gilbert may be preferred (Maxam and Gilbert, 1980, Meth. Enz.65:499; Ausubel et al., supra, Ch. 7).

The 5′ untranslated sequences of hTRT or other TRT mRNAs can bedetermined directly by cloning a “full-length” hTRT or other cDNA usingstandard methods such as reverse transcription of mRNA, followed bycloning and sequencing the resulting cDNA. Preferred oligo(dT)-primedlibraries for screening or amplifying full length cDNAs that have beensize-selected to include larger cDNAs may be preferred. Random primedlibraries are also suitable and often include a larger proportion ofclones that contain the 5′ regions of genes. Other well known methodsfor obtaining 5′ RNA sequences, such as the RACE protocol described byFrohman et al., 1988, Proc. Nat. Acad. Sci USA 85:8998, may also beused. If desired, the transcription start site of an hTRT or other TRTmRNA can be determined by routine methods using the nucleic acidsprovided herein (e.g., having a sequence of SEQ ID NO:1). One method isS1 nuclease analysis (Ausubel et al., supra) using a labeled DNA havinga sequence from the 5′ region of SEQ ID NO:1.

2) Chemical Synthesis of Nucleic Acids

The present invention also provides hTRT polynucleotides (RNA, DNA ormodified) that are produced by direct chemical synthesis. Chemicalsynthesis is generally preferred for the production of oligonucleotidesor for oligonucleotides and polynucleotides containing nonstandardnucleotides (e.g., probes, primers and antisense oligonucleotides).Direct chemical synthesis of nucleic acids can be accomplished bymethods known in the art, such as the phosphotriester method of Naranget al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brownet al., Meth. Enzymol. 68:109 (1979); the diethylphosphoramidite methodof Beaucage et al., Tetra. Lett., 22:1859 (1981); and the solid supportmethod of U.S. Pat. No. 4,458,066. Chemical synthesis typically producesa single stranded oligonucleotide, which may be converted into doublestranded DNA by hybridization with a complementary sequence, or bypolymerization with a DNA polymerase and an oligonucleotide primer usingthe single strand as a template. One of skill will recognize that whilechemical synthesis of DNA is often limited to sequences of about 100 or150 bases, longer sequences may be obtained by the ligation of shortersequences or by more elaborate synthetic methods.

It will be appreciated that the hTRT (or hTR or other) polynucleotidesand oligonucleotides of the invention can be made using nonstandardbases (e.g., other than adenine, cytidine, guanine, thymine, anduridine) or nonstandard backbone structures to provides desirableproperties (e.g., increased nuclease-resistance, tighter-binding,stability or a desired T_(M)). Techniques for rendering oligonucleotidesnuclease-resistant include those described in PCT publication WO94/12633. A wide variety of useful modified oligonucleotides may beproduced, including oligonucleotides having a peptide-nucleic acid (PNA)backbone (Nielsen et al., 1991, Science 254:1497) or incorporating2′-O-methyl ribonucleotides, phosphorothioate nucleotides, methylphosphonate nucleotides, phosphotriester nucleotides, phosphorothioatenucleotides, phosphoramidates. Still other useful oligonucleotides maycontain alkyl and halogen-substituted sugar moieties comprising one ofthe following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃OCH₃,OCH₃O(CH₂)_(n)CH₃, O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃ where n is from 1 toabout 10; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl oraralkyl; Cl; Br; CN; CF₃; OCF₃; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a cholesteryl group; a folate group; a reportergroup; an intercalator; a group for improving the pharmacokineticproperties of an oligonucleotide; or a group for improving thepharmacodynamic properties of an oligonucleotide and other substituentshaving similar properties. Folate, cholesterol or other groups whichfacilitate oligonucleotide uptake, such as lipid analogs, may beconjugated directly or via a linker at the 2′ position of any nucleosideor at the 3′ or 5′ position of the 3′-terminal or 5′-terminalnucleoside, respectively. One or more such conjugates may be used.Oligonucleotides may also have sugar mimetics such as cyclobutyls inplace of the pentofuranosyl group. Other embodiments may include atleast one modified base form or “universal base” such as inosine, orinclusion of other nonstandard bases such as queosine and wybutosine aswell as acetyl-, methyl-, thio- and similarly modified forms of adenine,cytidine, guanine, thymine, and uridine which are not as easilyrecognized by endogenous endonucleases. The invention further providesoligonucleotides having backbone analogues such as phosphodiester,phosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal,methylene(methylimino), 3′-N-carbamate, morpholino carbamate,chiral-methyl phosphonates, nucleotides with short chain alkyl orcycloalkyl intersugar linkages, short chain heteroatomic or heterocyclicintersugar (“backbone”) linkages, or CH₂—NH—O—CH₂, CH₂—N(CH₃)—OCH₂,CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones(where phosphodiester is O—P—O—CH₂), or mixtures of the same. Alsouseful are oligonucleotides having morpholino backbone structures (U.S.Pat. No. 5,034,506).

Useful references include Oligonucleotides and Analogues, A PracticalApproach, edited by F. Eckstein, IRL Press at Oxford University Press(1991); Antisense Strategies, Annals of the New York Academy ofSciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan etal., Jul. 9, 1993, J. Med. Chem. 36(14):1923-1937; Antisense Researchand Applications (1993, CRC Press), in its entirety and specificallyChapter 15, by Sanghvi, entitled “Heterocyclic base modifications innucleic acids and their applications in antisense oligonucleotides.”Antisense Therapeutics, ed. Sudhir Agrawal (Humana Press, Totowa, N.J.,1996).

D) Labeling Nucleic Acids

It is often useful to label the nucleic acids of the invention, forexample, when the hTRT or other oligonucleotides or polynucleotides areto be used as nucleic acid probes. The labels (see infra) may beincorporated by any of a number of means well known to those of skill inthe art. In one embodiment, an unamplified nucleic acid (e.g., mRNA,polyA mRNA, cDNA) is labeled. Means of producing labeled nucleic acidsare well known to those of skill in the art and include, for example,nick-translation, random primer labeling, end-labeling (e.g. using akinase), and chemical conjugation (e.g., photobiotinylation) orsynthesis. In another embodiment, the label is simultaneouslyincorporated during an amplification step in the preparation of thesample nucleic acids. Thus, for example, polymerase chain reaction (PCR)or other nucleic acid amplification method with labeled primers orlabeled nucleotides will provide a labeled amplification product. Inanother embodiment, transcription amplification using a labelednucleotide (e.g. fluorescein-labeled UTP and/or CTP) incorporates alabel into the transcribed nucleic acids. An amplification product mayalso, or alternatively, be labeled after the amplification is completed.

E) Illustrative Oligonucleotides

As noted supra and discussed in detail infra, oligonucleotides are usedfor a variety of uses including as primers, probes, therapeutic or otherantisense oligonucleotides, triplex oligonucleotides, and numerous otheruses as apparent from this disclosure. Table 2 provides certainillustrative specific oligonucleotides that may be used in the practiceof the invention. It will be appreciated that numerous other usefuloligonucleotides of the invention may be synthesized by one of skill,following the guidance provided herein.

In Table 2, “seq” means that the primer has been used, or is useful, forsequencing; “PCR” means that the primer has been used, or is useful, forPCR; “AS” means that means that the primer has been used, or is usefulfor antisense inhibition of telomerase activity; “CL” means that theprimer has been used, or is useful in cloning regions of hTRT genes orRNA, “mut” means that the primer has been used, or is useful forconstructing mutants of hTRT genes or gene products. “UC,” means “uppercase,” and “lc” means “lower case.” Mismatches and insertions (relativeto SEQ ID NO:1) are indicated by underlining; deletions are indicated bya “−”. It will be appreciated that nothing in Table 2 is intended tolimit the use of any particular oligonucleotide to any single use or setof uses.

TABLE 2 USEFUL OLIGONUCLEOTIDES USE primer 5′-sequence-3′ * Notesmismatch? * seq PCR AS CL MUT SEQ ID TCP1.1 GTGAAGGCACTGTTCAGCG x x 377TCP1.2 GTGGATGATTTCTTGTTGG x X 381 TCP1.4 CTGGACACTCAGCCCTTGG x X 382TCP1.5 GGCAGGTGTGCTGGACACT x x 383 TCP1.6 TTTGATGATGCTGGCGATG x x 384TCP1.7 GGGGCTCGTCTTCTACAGG Y x x 385 TCP1.8 CAGCAGGAGGATCTTGTAG x x 386TCP1.9 TGACCCCAGGAGTGGCACG x x 387 TCP1.10 TCAAGCTGACTCGACACCG x x 388TCP1.11 CGGCGTGACAGGGCTGC x x 389 TCP1.12 GCTGAAGGCTGAGTGTCC x x 390TCP1.13 TAGTCCATGTTCACAATCG x x 391 TCP1.14 CTGTGCTGGGCCTGGACGATA x x379 TCP1.15 CGCGTGGGTGAGGTGAGGTG x x 378 TCP1.16 TTTCCGTGTTGAGTGTTTC x x392 TCP1.17 GTCACCGTGTTGGGCAGG x x 393 TCP1.19 GCTACCTGCCCAACACGG x x394 TCP1.20 GCGCGAAGAACGTGCTGG x x 395 TCP1.21 CA-CTGCTCCTTGTCGCCTG Y xx 396 TCP1.22 TTCCCAAGGACTTTGTTGC x x 397 TCP1.24 TGTTCCTCAAGACGCACTG Yx x 398 TCP1.25 TACTGCGTGCGTCGGTATG x x 399 TCP1.26 GGTCTTGCGGCTGAAGTGTx x 400 TCP1.27 TGGTTCACCTGCTGGCACG x x 40) TCP1.28 GTGGTTTCTGTGTGGTGTCx x 402 TCP1.29 GACACCACACAGAAACCAC x x 403 TCP1.30 GTGCCAGCAGGTGAACCAGx x 404 TCP1.32B GCAGTGCGTCTTGAGGAGC x x 405 TCP1.33TGGAACCATAGCGTCAGGGAG x x 406 TCP1.34 GGCCTCCCTGACGCTATGGTT x x 407TCP1.35 GC(GT)CGGCGCTGCCACTCAGG x x 408 TCP1.35t GCTCGGCGCTGCCACTCAGG409 TCP1.36 ACGCCGAGACCAAGCACTTC x x 410 TCP1.38 CCAAAGAGGTGGCTTCTTCG xx 4)1 TCP1.39 AAGGCCAGCACGTTCTTCGC x x 412 TCP1.40 CACGTTCGTGCGGCGCCTG xx 413 TCP1.41 CCTTCACCACCAGCGTGCG x x 414 TCP1.42 GGCGACGACGTGCTGGTTC xx 415 TCP1.43 GGCTCAGGGGCAGCGCCAC x x 416 TCP1.44 CTGGCAGGTGTACGGCTTC xx 417 TCP1.45 GCGTGGACCGAGTGACCGTGGTTTC x x 418 TCP1.46GACGTGGTGGCCGCGATGTGG x x 419 TCP1.47 GAAGTCTGCCGTTGCCCAAGAG x x 420TCP1.48 GACACCACACAGAAACCACGGTCAC x x 421 TCP1.49 CGCCCCCTCCTTCCGCCAGGTx x 422 TCP1.50 CGAAGCCGAAGGCCAGCACGTTCTT x x 423 TCP1.51GGTGGCCCGAGTGCTGCAGAGG x x 424 TCP1.52 GTAGCTGCGCACGCTGGTGGTGAAG x x 425TCP1.53 TGGGCGACGACGTGCTGGTTCA x x 426 TCP1.54 TATGGTTCCAGGCCCGTTCGCATCCx x 427 TCP1.55 CCAGCTGCGCCTACCAGGTGTGC x x 428 TCP1.56GGCCTCCCTGACGCTATGGTTCCAG x x 429 TCP1.57 GGTGCTGCCGCTGGCCACGTTCG x x430 TCP1.58 TCCCAGGGCACGCACACCAGGCACT x x 431 TCP1.59GTACAGGGCACACCTTTGGTCACTC x x 432 TCP1.60 TCGACGACGTACACACTCATCAGCC x x433 TCP1.61 AGCGGCAGCACCTCGCGGTAGTGGC x x 434 TCP1.62CCACCAGCTCCTTCAGGCAGGACAC x x 435 TCP1.63 CCAGGGCTTCCCACGTGCGCAGCAG x x436 TCP1:64 CGCACGAACGTGGCCAGCGGCAGCA x x 437 TCP1.65TGACCGTGGTTTCTGTGTGGTGT x x 438 TCP1.66 CCCTCTTCAAGTGCTGTCTGATTCC x x439 TCP1.67 ATCGCGGCCACCACGTCCCT x x 440 TCP1.68TGCTCCAGACACTCGGCCGGTAGAA x x 441 TCP1.69 ACGAAGCCGTACACCTGCC x x 442TCP1.72 CGACATCCCTGCGTTCTTGGCTTTC x x 443 TCP1.73 CACTGCTGGCCTCATTCAGGGx x 444 TCP1.74 GCGACATGGAGAACAAGC x x 445 TCP1.75 GCAGCCATACTTCAGGGACACx x 446 TCP1.76 CCATCCTCTCCACGCTGCTC x x 447 TCP1.77GCGATGACCTCCGTGAGCCTG x x 448 TCP1.78 CCCAGGACAGGCTCACGGA x x 449billTCP1 CCTCTTCAAGTGCTGTCTGATTCC x x 450 billTCP2CAGCTCGACGACGTACACACTCATC x x 451 billTCP4 CTGACGTCCAGACTCCGCTTCAT x x452 billTCP6 AGCTTGTTCTCCATGTCGCCGTAG x x 380 rpprim0lGACCTGAGCAGCTCGACGACGTACACAC x x 453 TCATC Lt1 GTCGTCGAGCTGCTCAGGTC x x454 Lt2 AGCACGCTGAACAGTGCCTT x x 455 Lt3 GACCTGAGCAGCTCGACGAC x x 456Lt4 AAGGCACTGTTCAGCGTGCT x x 457 Lt5 CGGCCGAGTGTCTGGAGCAA Y x x 458 Lt6GGATGAAGCGGAGTCTGGA x x 459 BamHI Lt7 ATGGATCCGTCGTCGAGCTGCTCAGGTCTBamHI Site Y x x 460 Salt Lt8 ATCAGC Pvu II site (not Sal I) Y x x 461TGAGCACGCTGAACAGTGCCTTC K303 GTCTCCGTGACATAAAAGAAAGAC x x 462 K304GCCAAGTTCCTGCACTGGCT x x 463 K305 GCCTGTTCTTITGAAACGTGGTCT x x 464 K306XXGCCTGTTCTTTTGAAACGTGGTCT X = biotin, = K305 x x 465 K311GTCAAGATGCCTGAGATAGAAC x x 466 K312 TGCTTAGCTTGTGGGGGTGTCA x x 467 K313TGCTTAGCTTGTGGGGGTGTCA x x 467 K320 GCTGCGTCCTGCTGCGCACGT x x 468 K321CAGCGGGGAGCGCGCGGCATC x x 469 K322 TGGGCCACCAGCGCGCGGAAA x x 470slanti.1 CGGCGCAGCCCGTCAGGCTTGGGG Y x x 471 slanti.2 CCGACAGCTCCCGCAGCTGCACCC Y x x 472 slanti.3 CGTACACACTCATCAGCCAGTGCAGGAA x x473 CTTGGC slanti.4 CGCGCCCGCTCGTAGTTGAGCACGCTGA x x 474 ACAGTGCCTTCACCCTCG slanti.5 GCGGAGTCTGGACGTCAGCAGGGCGGGC x x 475 CTGGCTTCCCG UTR2ATTTGACCCACAGGGACCCCCATCCAG x x 476 FW5 ATGACCGCCCTCCTCGTGAG x x 477Nam1 GCCACCCCCGCGATGCC x x 478 Nam2 AGCCCTGGCCCCGGCCA x x 479 Nam3TCCCACGTGCGCAGCAG x x 480 Nam4 AGCAGGACGCAGCGCTG x x 481 PE01CGCGGTAGTGGCTGCGCAGCAGGGAGCG x x 482 CACGGC PE02CCAGGGCTTCCCACGTGCGCAGCAGGAC x x 483 GCAGCGC LM101 CTAGTCTAGATCA / Xba Isite / HA tag / hTRT x 484 GCTAGCGTAATCTGGAACATCGTA into pGRN121 TGGGTA/ GTCCAGGATGGTCTTGAAGTC LM103 TACCATGGGCTACCCATACGACGTTCCAG inserts HAtag into a Nde I site x 485 ATTACGCTCA at 5′ end of hTRT LM104TATGAGCGTAATCTGGAACGTCGTATGGG anneals to LM103 x 486 TAGCCCATGG LM105GTGTACGTCGTCGAGCTCCTCAGGTCTGC change = F560A x 487 CTTTT (phe > ala)ATGTCACGGAG LM106 GTGTACGTCGTCGAGCTCCTCAGGTCTTT change = F561A x 488CGCTTATGTC (phe > ala) ACGGAGACC LM107 CCTCAGGTCTTTCTTTTGCTGTCACGGAGAchange = Y562A x 489 CAACGTTT (tyr > ala) CAAAAGAACAG LM108GGTCTTTCTTTTATGTCGCGGAGACAACG change = T564A x 490 TTT (tyr > ala)CAAAAGAACAG LM109 CTTTCTTTTATGTCACGGCGACAACGTTT change = E565A x 491CAAAAGAACA G LM_FFYTE ATGAGTGTGTACGTCGTCGAGCTCCTCAG deletion of FFYVTE x492 GTCTACCACG (aa560-565) TTTCAAAAGAACAGGCTCTTTTTC TCP061:GGCTGATGAGTGTGTACGTCGTCGA complement to TCP1.61 x x 493 HUMO1:ACGTGGTCTCCGTGACATAAAAGAA to DD motif, designed to possibly anneal tomTRT x x x 494 HUMO2: AGGTCTTTCTTTTATGTCACGGA to DD motif, designed topossibly anneal to mTRT x x x 495 HUMO3: CACAGACCCCCGTCGCCTGGTC designedto possibly anneal to mTRT x x x 496 HUMO4: CGGAGTCTGGACGTCAGCAGGGCdesigned to possibly anneal to mTRT x x x 497 SLW F1NcgcggatccgtaactaaaATGCCGCGCGCTCCCCG for GST fusion construct (782 to1636) x x 498 CTGC UC = hTRT seq, lc = Bam HI site + 2 stop codons SLWF1C ccggaattcgttagttacttaCAAAGAGGTGGCTTCTT for GST fusion construct (782to 1636) x x 499 CGGC UC = hTRT seq, lc = EcoR I site + 3 stop codonsSLW F1N / SLW F1C amplify a 893 nt piece of pGRN121 (782 to 1636) SLWF2N cgcggatccgtaactaaaGCCACCTCTTTGGAGGGTGCG for GST fusion construct(1625 to 2458) x x 500 UC = hTRT seq, lc = EcoR I site + 3 stop codonsSLW F2C ccgtaattctttagttacttaGACCTGAGCAGCTCGACGAC for GST fusionconstruct (1625 to 2458) x x 501 UC = hTRT seq, lc = EcoR I site + 3stop codons SLW F2N / SLW F2C amplify a 872 nt piece of pGRN121 (1625 to2458) SLW F3N cgcggatccgtaactaaaATGAGTGTGTACGTCGTCGAG for GST fusionconstruct (2426 to 3274) x x 502 UC = hTRT seq, lc = Bam HI site + 2stop codons SLW F3C ccttgaattcgttagttacttaGATCCCCTGGCACTGGACG for GSTfusion construct (2426 to 3274) x x 503 UC = hTRT seq, lc = EcoR Isite + 3 stop codons SLW F3N / SLW F3C amplify a 887 nt piece of pGRN121(2426 to 3274) SLW F4N cgcggatccgtaactaaaaATCCCGCAGGGCTCCATCCTC for GSTfusion construct (3272 to 4177) x x 504 UC = hTRT seq, lc = Bam HIsite + 2 stop codons SLW F4C ccggaattcgttattttacttaGTCCAGGATGGTCTTGAAGTCfor GST fusion construct (3272 to 4177) x x 505 UC = hTRT seq, lc = EcoRI site + 3 stop codons SLW F4N / SLW F4C amplify a 944 nt piece ofpGRN121 (3272 to 4177) 40-60 GGCATCGCGGGGGTGGCCGGG phosphorothioatc x506 260-280 GGACACCTGGCGGAAGGAGGG phosphorothioatc x 507 500-520GCGTGCCAGCAGGTGAACCAG phosphorothioatc x 508 770-790CTCAGGGGCAGCGCCACGCCT phosphorothioate x 509 885-905AGGTGGCTTCTTCGGCGGGTC phosphorothioate x 510 1000-1020GGACAAGGCGTGTCCCAGGGA phosphorothioate x 5I1 1300-1320GCTGGGGTGACCGCAGCTCGC phosphorothioate x 512 1520-1540GATGAACTTCTTGGTGTTCCT phosphorothioate x 513 2110-2130GTGCGCCAGGCCCTGTGGATA phosphorothioate x 514 2295-2315GCCCATGGGCGGCCTTCTGGA phosphorothioate x 515 2450-2470GAGGCCACTGCTGGCCTCATT phosphorothioate x 516 2670-2690GGGTGAGGTGAGGTGTCACCA phosphorothioate x 517 3080-3110GCTGCAGCACACATGCGTGAAACCTGTACGC phosphorothioate x 518 3140-3160GACGCGCAGGAAAAATGTGGG phosphorothioate x 519 3690-3710CCGAGCGCCAGCCTGTGGGGA phosphorothioate x 520 55-75 CAGCGGGGAGCGCGCGGCATCphosphorothioatc x 521 151-171 CAGCACCTCGCGGTAGTGGCT phosphorothioate x522 TP1.1 TCAAGCCAAACCTGAATCTGAG x 523 TP1.2 CCCGAGTGAATCTTTCTACGC x 524TP1.3 GTCTCTGGCAGTTTCCTCATCCC x 525 TP1.4 TTTAGGCATCCTCCCAAGCACA x 526

IV. TRT Proteins and Peptides

A) Generally

The invention provides a wide variety of hTRT proteins useful for, interalia, production of telomerase activity, inhibition of telomeraseactivity in a cell, induction of an anti-hTRT immune response, as atherapeutic reagent, as a standard or control in a diagnostic assay, asa target in a screen for compounds capable of activation or inhibitionof an activity of hTRT or telomerase, and numerous other uses that willbe apparent to one of skill or are otherwise described herein. The hTRTof the invention include functionally active proteins (useful for e.g.,conferring telomerase activity in a telomerase-negative cell) andvariants, inactive variants (useful for e.g., inhibiting telomeraseactivity in a cell), hTRT polypeptides, and telomerase RNPs (e.g.,ribonucleoprotein complexes comprising the proteins) that exhibit one,several, or all of the functional activities of naturally occurring hTRTand telomerase, as discussed in greater detail for illustrativepurposes, below.

In one embodiment, the hTRT protein of the invention is a polypeptidehaving a sequence as set forth in FIG. 17 (SEQ ID NO:2), or a fragmentthereof. In another embodiment, the hTRT polypeptide differs from SEQ IDNO:2 by internal deletions, insertions, or conservative substitutions ofamino acid residues. In a related embodiment, the invention provideshTRT polypeptides with substantial similarity to SEQ ID NO:2. Theinvention further provides hTRT polypeptides that are modified, relativeto the amino acid sequence of SEQ ID NO:2, in some manner, e.g.,truncated, mutated, derivatized, or fused to other sequences (e.g., toform a fusion protein). Moreover, the present invention providestelomerase RNPs comprising an hTRT protein of the invention complexedwith a template RNA (e.g., hTR). In other embodiments, one or moretelomerase-associated proteins is associated with hTRT protein and/orhTR.

The invention also provides other naturally occurring hTRT species ornonnaturally occurring variants, such as proteins having the sequenceof, or substantial similarity to SEQ ID NO:5 [[FIG. 20], SEQ ID NO:10[FIG. 19], and fragments, variants, or derivatives thereof.

The invention provides still other hTRT species and variants. Oneexample of an hTRT variant may result from ribosome frameshifting ofmRNA encoded by the clone 712562 (SEQ ID NO:3 [FIG. 18]) or the pro90variant hTRT shown in SEQ ID NO:4 [FIG. 20] and so result in thesynthesis of hTRT polypeptides containing all the TRT motifs (for ageneral example, see, e.g., Tsuchihashi et al., 1990, Proc. Natl. Acad.Sci. USA 87:2516; Craigengen et al., 1987, Cell 50:1; Weiss, 1990, Cell62:117). Ribosome frameshifting can occur when specific mRNA sequencesor secondary structures cause the ribosome to “stall” and jump onenucleotide forwards or back in the sequence. Thus, a ribosome frameshiftevent on the 712562 mRNA could cause the synthesis of an approximately523 amino acid residue polypeptide. A ribosome frameshift event on thepro90 sequence could result in a protein with approximately 1071residues. It will be appreciated that proteins resulting from ribosomeframeshifting can also be expressed by synthetic or recombinanttechniques provided by the invention.

Human TRT proteins, peptides, and functionally equivalent proteins maybe obtained by purification, chemical synthesis, or recombinantproduction, as discussed in greater detail below.

B) TRT Protein Activities

The TRT polypeptides of the invention (including fragments, variants,products of alternative alleles, and fusion proteins) can have one ormore, or all of the functional activities associated with native hTRT.Except as noted, as used herein, an hTRT or other TRT polypeptide isconsidered to have a specified activity if the activity is exhibited byeither the hTRT protein without an associated RNA (e.g., hTR) or in anhTRT-associated RNA (e.g., hTR) complex. The hTR-binding activity ofhTRT is one example of an activity associated with the hTRT protein.Methods for producing complexes of nucleic acids (e.g., hTR) and thehTRT polypeptides of the invention are described infra.

Modification of the hTRT protein (e.g., by chemical or recombinantmeans, including mutation or modification of a polynucleotide encodingthe hTRT polypeptide or chemical synthesis of a polynucleotide that hasa sequence different than a native polynucleotide sequence) to have adifferent complement of activities than native hTRT can be useful intherapeutic applications or in screening for specific modulators of hTRTor telomerase activity. In addition, assays for various hTRT activitiescan be particularly useful for identification of agents (e.g., activitymodulating agents) that interact with hTRT or telomerase to changetelomerase activity.

The activities of native hTRT, as discussed infra, include telomerasecatalytic activity (which may be either processive or non-processiveactivity); telomerase processivity; conventional reverse transcriptaseactivity; nucleolytic activity; primer or substrate (telomere orsynthetic telomerase substrate or primer) binding activity; dNTP bindingactivity; RNA (i.e., hTR) binding activity; and protein binding activity(e.g., binding to telomerase-associated proteins, telomere-bindingproteins, or to a protein-telomeric DNA complex). It will be understood,however, that present invention also provides hTRT compositions withoutany particular hTRT activity but with some useful activity related tothe hTRT or other TRT proteins (e.g., certain typically shortimmunogenic peptides, inhibitory peptides).

1) Telomerase Catalytic Activity

As used herein, a polypeptide of the invention has “telomerase catalyticactivity,” when the polypeptide is capable of extending a DNA primerthat functions as a telomerase substrate by adding a partial, one, ormore than one repeat of a sequence (e.g., TTAGGG) encoded by a templatenucleic acid (e.g., hTR). This activity may be processive ornonprocessive. Processive activity occurs when a telomerase RNP addsmultiple repeats to a primer or telomerase before the DNA is released bythe enzyme complex. Non-processive activity occurs when telomerase addsa partial, or only one, repeat to a primer and is then released. Invivo, however, a non-processive reaction could add multiple repeats bysuccessive rounds of association, extension, and dissociation. This canoccur in vitro as well, but it is not typically observed in standardassays due to the vastly large molar excess of primer over telomerase instandard assay conditions.

To characterize an hTRT polypeptide as having non-processive activity, aconventional telomerase reaction is performed using conditions thatfavor a non-processive reaction, for example high temperatures (i.e.,35-40 EC, typically 37 EC), low dGTP concentrations (1 μM or less), highprimer concentrations (5 μM or higher), and high dATP/TTP concentrations(2 mM or higher), with the temperature and dGTP typically having thegreatest effect. To characterize an hTRT polypeptide as havingprocessive activity, a conventional telomerase reaction is performedusing conditions that favor a processive reaction (for example, 27-34°C., typically 30° C.), high dGTP concentration (10 μM or higher), lowprimer concentration (1 μM or lower), and/or low dATP and TTPconcentrations (0.3-1 mM) with temperature and dGTP typicallyconcentration being the most critical. Alternatively, a TRAP assay (forprocessive or moderately processive activity) or the dot-blot and gelblot assays (for processive activity) may be used. The hTRT polypeptideof the invention can possess a non-processive activity, but not aprocessive activity (e.g., if an alteration of the hTRT polypeptidereduces or eliminates the ability to translocate), can be solelyprocessive, or can possess both activities.

a) Non-processive Activity

A non-processive telomerase catalytic activity can extend the DNA primerfrom the position where the 3′ end anneals to the RNA template to the 5′end of the template sequence, typically terminating with the addition ofthe first G residue (as, for example, when the template is hTR). Asshown below, the exact number of nucleotides added is dependent on theposition of the 3′ terminal nucleotide of the primer in the TTAGGGrepeat sequence.

Nonprocessive Activity

i) - - - TTAGGGttag (DNA)

3′ - - - AUCCCAAUC - - - 5′ (RNA) SEQ ID NO:527

ii) - - - TTAGggttag (DNA)

3′ - - - AUCCCAAUC - - - 5′ (RNA) SEQ ID NO:527

In DNA, UC,=primer, lc=added nucleotides

Thus, 4 nucleotides are added to the - - - TTAGGG primer (i) while 6nucleotides are added to the - - - TTAG primer (ii). The first repeatadded by telomerase in a processive reaction is equivalent to this step;however, in a processive reaction telomerase performs a translocationstep where the 3′ end is released and re-bound at the 3′ region of thetemplate in a position sufficient to prime addition of another repeat(see Morin, 1997, Eur. J. Cancer 33:750).

A fully non-processive reaction produces only one band in a conventionalassay using a single synthetic primer. Because this result could also beproduced by other enzymes, such as a terminal transferase activity, itmay be desirable in some applications to verify that the product is aresult of a telomerase catalytic activity. A telomerase (comprisinghTRT) generated band can be distinguished by several additionalcharacteristics. The number of nucleotides added to the end of theprimer should be consistent with the position of the primer 3′ end.Thus, a - - - TTAGGG primer should have 4 nucleotides added and a - - -TTAG primer should have 6 nucleotides added (see above). In practice,two or more sequence permuted primers can be used which have the sameoverall length but different 5′ and 3′ endpoints. As an illustrativeexample, the non-processive extension of primers 5′-TTAGGGTTAGGGTTAGGG(SEQ ID NO:528) and 5′-GTTAGGGTTAGGGTTAGG (SEQ ID NO:529) will generateproducts whose absolute length will be one nucleotide different (4 addedto 5′-TTAGGGTTAGGGTTAGGG (SEQ ID NO:528) for a 22 nt total length, and 5added to 5′-GTTAGGGTTAGGGTTAGG (SEQ ID NO:529) for a 23 nt totallength). The nucleotide dependence of the reaction should be consistentwith the position of the primer terminus. Thus, a - - - TTAGGG primerproduct should require dGTP, TTP, and dATP, but not dCTP, and a - - -AGGGTT primer product should require dGTP and DATP, but not TTP or dCTP.The activity should be sensitive to RNAase or micrococcal nucleasepre-treatment (see Morin, 1989, Cell 59: 521) under conditions that willdegrade hTR and so eliminate the template.

b) Processive Activity

In practice, a processive activity is easily observed by the appearanceof a six nucleotide ladder in a conventional assay, TRAP assay, orgel-blot assay. A dot-blot assay can also be used, but no ladder isdetected in such a method. The conventional assay is described in Morin,1989, Cell 59:521, which is incorporated herein in its entirety and forall purposes. The TRAP assay is described in U.S. Pat. No. 5,629,154;see also, PCT publication WO 97/15687, PCT publication WO 95/13381;Krupp et al. Nucleic Acids Res., 1997, 25: 919; and Wright et al., 1995,Nuc. Acids Res. 23:3794, each of which is incorporated herein in itsentirety and for all purposes. The dot blot immunoassay is described indetail in co-pending U.S. patent application Ser. No. 08/833,377, filedApr. 14, 1997, which is incorporated herein by reference in its entiretyand for all purposes. The dot blot assay can be used in a format inwhich a non-processive activity, which does not add the 3 or morerepeats required for stable hybridization of the (CCCUAA)n probe used todetect the activity, is tested with compounds or hTRT variants todetermine if the same generates processivity, i.e., if the probe detectsan expected telomerase substrate, then the compound or mutant is able tochange the non-processive activity to a processive activity. Otherassays for processive telomerase catalytic activity can also be used,e.g., the stretch PCR assay of Tatematsu et al., 1996, Oncogene 13:2265.The gel-blot assay, a combination of the conventional and dot blotassays can also be used. In this variation a conventional assay isperformed with no radiolabeled nucleotide and with high dGTPconcentrations (e.g., 0.1-2 mM). After performing the conventionalassay, the synthesized DNA is separated by denaturing PAGE andtransferred to a membrane (e.g., nitrocellulose). Telomeric DNA (theproduct of telomerase—an extended telomerase primer or substrate) canthen be detected by methods such as hybridization using labeledtelomeric DNA probes (e.g., probes containing the CCCTAA sequence, asused in the dot blot assay, supra). An advantage of this technique isthat it is more sensitive than the conventional assay and providesinformation about the size of the synthesized fragments and processivityof the reaction.

c) Activity Determinations

The telomerase activity of an hTRT polypeptide can be determined usingan unpurified, partially purified or substantially purified hTRTpolypeptide (e.g., in association with hTR), in vitro, or afterexpression in vivo. For example, telomerase activity in a cell (e.g., acell expressing a recombinant hTRT polypeptide of the invention) can beassayed by detecting an increase or decrease in the length of telomeres.Typically assays for telomerase catalytic activity are carried out usingan hTRT complexed with hTR; however, alternative telomerase templateRNAs may be substituted, or one may conduct assays to measure anotheractivity, such as telomerase-primer binding. Assays to determine thelength of telomeres are known in the art and include hybridization ofprobes to telomeric DNA (an amplification step can be included) and TRFanalysis i.e., the analysis of telomeric DNA restriction fragments[TRFs] following restriction endonuclease digestion, see PCTpublications WO 93/23572 and WO 96/41016; Counter et al., 1992, EMBO J.11:1921; Allsopp et al., 1992, Proc. Nat'l. Acad. Sci. USA 89:10114;Sanno, 1996, Am J Clin Pathol 106:16 and Sanno, 1997, Neuroendocrinology65:299.

The telomerase catalytic activity of an hTRT polypeptide may bedetermined in a number of ways using the assays supra and othertelomerase catalytic activity assays. According to one method, the hTRTprotein is expressed (e.g., as described infra) in a telomerase negativehuman cell in which hTR is expressed (i.e., either normally in the cellor through recombinant expression), and the presence or absence oftelomerase activity in the cell or cell lysate is determined. Examplesof suitable telomerase-negative cells are IMR 90 (ATCC, #CCL-186) or BJcells (human foreskin fibroblast line; see, e.g., Feng et al., 1995,Science 269:1236). Other examples include retinal pigmented epithelialcells (RPE), human umbilical vein endothelial cells (HUVEC; ATCC#CRL-1730), human aortic endothelial cells (HAEC; Clonetics Corp,#CC-2535), and human mammary epithelial cells (HME; Hammond et al.,1984, Proc. Nat'l. Acad. Sci. USA 81:5435; Stampfer, 1985, J. TissueCulture Methods 9:107). In an alternative embodiment, the hTRTpolypeptide is expressed (e.g., by transfection with an hTRT expressionvector) in a telomerase positive cell, and an increase in telomeraseactivity in the cell compared to an untransfected control cell isdetected if the polypeptide has telomerase catalytic activity. Usuallythe telomerase catalytic activity in a cell transfected with a suitableexpression vector expressing hTRT will be significantly increased, suchas at least about 2-fold, at least about 5-fold, or even at least about10-fold to 100-fold or even 1000-fold higher than in untransfected(control) cells.

In an alternative embodiment, the hTRT protein is expressed in a cell(e.g., a telomerase negative cell in which hTR is expressed) as a fusionprotein (see infra) having a label or an “epitope tag” to aid inpurification. In one embodiment, the RNP is recovered from the cellusing an antibody that specifically recognizes the tag. Preferred tagsare typically short or small and may include a cleavage site or otherproperty that allows the tag to be removed from the hTRT polypeptide.Examples of suitable tags include the Xpress™ epitope (Invitrogen, Inc.,San Diego Calif.), and other moieties that can be specifically bound byan antibody or nucleic acid or other equivalent method such as thosedescribed in Example 6. Alternative tags include those encoded bysequences inserted, e.g., into SEQ ID NO:1 upstream of the ATG codonthat initiates translation of the protein of SEQ ID NO:2, which mayinclude insertion of a (new) methionine initiation codon into theupstream sequence.

It will be appreciated that when an hTRT variant is expressed in a cell(e.g., as a fusion protein) and subsequently isolated (e.g., as aribonucleoprotein complex), other cell proteins (i.e.,telomerase-associated proteins) may be associated with (directly orindirectly bound to) the isolated complex. In such cases, it willsometimes be desirable to assay telomerase activity for the complexcontaining hTRT, hTR and the associated proteins.

2) Other Telomerase or TRT Protein Activities

The hTRT polypeptides of the invention include variants that lacktelomerase catalytic activity but retain one or more other activities oftelomerase. These other activities and the methods of the invention formeasuring such activities include (but are not limited to) thosediscussed in the following sections.

a) Conventional Reverse Transcriptase Activity

Telomerase conventional reverse transcriptase activity is described in,e.g., Morin, 1997, supra, and Spence et al., 1995, Science 267:988.Because hTRT contains conserved amino acid motifs that are required forreverse transcriptase catalytic activity, hTRT has the ability totranscribe certain exogenous (e.g., non-hTR) RNAs. A conventional RTassay measures the ability of the enzyme to transcribe an RNA templateby extending an annealed DNA primer. Reverse transcriptase activity canbe measured in numerous ways known in the art, for example, bymonitoring the size increase of a labeled nucleic acid primer (e.g., RNAor DNA), or incorporation of a labeled dNTP. See, e.g., Ausubel et al.,supra.

Because hTRT specifically associates with hTR, it can be appreciatedthat the DNA primer/RNA template for a conventional RT assay can bemodified to have characteristics related to hTR and/or a telomeric DNAprimer. For example, the RNA can have the sequence (CCCTAA)_(n), where nis at least 1, or at least 3, or at least 10 or more (SEQ ID NO:530). Inone embodiment, the (CCCTAA)_(n) region is at or near the 5′ terminus ofthe RNA (similar to the 5′ locations of template regions in telomeraseRNAs). Similarly, the DNA primer may have a 3′ terminus that containsportions of the TTAGGG telomere sequence, for example X_(n)TTAG (SEQ IDNO:531), X_(n)AGGG (SEQ ID NO:532), X_(n)(TTAGGG)_(q)TTAG (SEQ IDNOS:533-536), etc., where X is a non-telomeric sequence and n is 8-20,or 6-30, and q is 1-4. In another embodiment, the DNA primer has a 5′terminus that is non-complementary to the RNA template, such that whenthe primer is annealed to the RNA, the 5′ terminus of the primer remainsunbound. Additional modifications of standard reverse transcriptionassays that may be applied to the methods of the invention are known inthe art.

b) Nucleolytic Activity

Telomerase nucleolytic activity is described in e.g., Morin, 1997,supra; Collins and Grieder, 1993, Genes and Development 7:1364.Telomerase possesses a nucleolytic activity (Joyce and Steitz, 1987,Trends Biochem. Sci. 12:288); however, telomerase activity has definingcharacteristics. Telomerase preferentially removes nucleotides, usuallyonly one, from the 3′ end of an oligonucleotide when the 3′ end of theDNA is positioned at the 5′ boundary of the DNA template sequence, inhumans and Tetrahymena, this nucleotide is the first G of the telomericrepeat (TTAGG in humans). Telomerase preferentially removes G residuesbut has nucleolytic activity against other nucleotides. This activitycan be monitored. Two different methods are described here forillustrative purposes. One method involves a conventional telomerasereaction with a primer that binds the entire template sequence (i.e.,terminating at the template boundary; 5′-TAGGGATTAG (SEQ ID NO:537) inhumans). Nucleolytic activity is observed by monitoring the replacementof the last dG residue with a radiolabeled dGTP provided in the assay.The replacement is monitored by the appearance of a band at the size ofthe starting primer as shown by gel electrophoresis and autoradiography.

A preferred method uses a DNA primer that has a “blocked” 3′ terminusthat cannot be extended by telomerase. The 3′-blocked primer can be usedin a standard telomerase assay but will not be extended unless the 3′nucleotide is removed by the nucleolytic activity of telomerase. Theadvantage of this method is that telomerase activity can be monitored byany of several standard means, and the signal is strong and easy toquantify. The blocking of the 3′ terminus of the primer can beaccomplished in several ways. One method is the addition of a3′-deoxy-dNTP residue at the 3′ terminus of the primer using standardoligonucleotide synthesis techniques. This terminus has a 2′ OH but notthe 3′ OH required for telomerase. Other means of blocking the 3′terminus exist, for instance, a 3′ dideoxy terminus, a 3′-amineterminus, and others. An example of a primer for an hTRT nucleolyticassay is 5′-TTAGGGTTAGGGTTA (G_(3′H)) (SEQ ID NO:538) where the lastresidue denotes a 3′-deoxy-guanosine residue (Glen Research, Sterling,Va.). Numerous other variations for a suitable primer based on thedisclosure are known to those of skill in the art.

c) Primer (Telomere) Binding Activity

Telomerase primer (telomere) binding activity is described in e.g.,Morin, 1997, supra; Collins et al., 1995, Cell 81:677; Harrington et al,1995, J. Biol. Chem. 270:8893. Telomerase is believed to have two siteswhich bind a telomeric DNA primer. The RT motifs associated with primerbinding indicate hTRT and/or hTRT/hTR possesses DNA primer bindingactivity. There are several ways of assaying primer binding activity;however, a step common to most methods is incubation of a labeled DNAprimer with hTRT or hTRT/hTR or other TRT/TR combinations underappropriate binding conditions. Also, most methods employ a means ofseparating unbound DNA from protein-bound DNA; those methods include thefollowing.

i) Gel-shift assays (also called electrophoretic/mobility shift assays)are those in which unbound DNA primer is separated from protein-boundDNA primer by electrophoresis on a nondenaturing gel (Ausubel et al.,supra).

ii) Matrix binding assays include several variations to the basictechnique, which involves binding the hTRT or hTRT/hTR complex to amatrix (e.g., nitrocellulose), either before or after incubation withthe labeled primer. By binding the hTRT to a matrix, the unbound primercan be mechanically separated from bound primer. Residual unbound DNAcan be removed by washing the membrane prior to quantitation. Those ofskill recognize there are several means of coupling proteins to suchmatrices, solid supports, and membranes, including chemical,photochemical, UV cross-linking, antibody/epitope, and non-covalent(hydrophobic, electrostatic, etc.) interactions.

The DNA primer can be any DNA with an affinity for telomerase, such as,for example, a telomeric DNA primer like (TTAGGG)_(n), where n could be1-10 and is typically 3-5 (SEQ ID NO:539). The 3′ and 5′ termini can endin any location of the repeat sequence. The primer can also have 5′ or3′ extensions of non-telomeric DNA that could facilitate labeling ordetection. The primer can also be derivatized, e.g., to facilitatedetection or isolation.

d) dNTP Binding Activity

Telomerase dNTP binding activity is described in e.g., Morin, 1997,supra; Spence et al., supra. Telomerase requires dNTPs to synthesizeDNA. The hTRT protein has a nucleotide binding activity and can beassayed for dNTP binding in a manner similar to other nucleotide bindingproteins (Kantrowitz et al., 1980, Trends Biochem. Sci. 5:124).Typically, binding of a labeled dNTP or dNTP analog can be monitored asis known in the art for non-telomerase RT proteins.

e) RNA (i.e., hTR) Binding Activity

Telomerase RNA (i.e., hTR) binding activity is described in e.g., Morin,1997, supra; Harrington et al., 1997, Science 275:973; Collins et al.,1995, Cell 81:677. The RNA binding activity of a TRT protein of theinvention may be assayed in a manner similar to the DNA primer bindingassay described supra, using a labeled RNA probe. Methods for separatingbound and unbound RNA and for detecting RNA are well known in the artand can be applied to the activity assays of the invention in a mannersimilar to that described for the DNA primer binding assay. The RNA canbe full length hTR, fragments of hTR or other RNAs demonstrated to havean affinity for telomerase or hTRT. See U.S. Pat. No. 5,583,016 and PCTPub. No. 96/40868.

3) Telomerase Motifs as Targets

The present invention, as noted supra, provides in addition torecombinant hTRT with a full complement (as described supra) ofactivities, hTRT polypeptides having less than the full complement ofthe telomerase activities of naturally occurring telomerase or hTRT orother TRT proteins. It will be appreciated that, in view of thedisclosure herein of the RT and telomerase-specific motifs of TRT,alteration or mutation of conserved amino acid residues, such as arefound in the motif sequences discussed supra, will result in loss-ofactivity mutants useful for therapeutic, drug screening andcharacterization, and other uses. For example, as described in Example1, deletion of motifs B through D in the RT domains of the endogenousTRT gene in S. pombe resulted in haploid cells in which telomereprogressively shortened to the point where hybridization of a telomereprobe to telomeric repeats became almost undetectable, indicating a lossof telomerase catalytic activity. Similarly, alterations in the WxGxS(SEQ ID NO:540) site of motif E can affect telomerase DNA primer bindingor function. Additionally, alterations of the amino acids in the motifsA, B′, and C can affect the catalytic activity of telomerase. Mutationof the DD motif of hTRT can significantly reduce or abolish telomeraseactivity (see Example 16).

C) Synthesis of HTRT and Other TRT Polypeptides

The invention provides a variety of methods for making the hTRT andother TRT polypeptides disclosed herein. In the following sections,chemical synthesis and recombinant expression of hTRT proteins,including fusion proteins, is described in some detail.

1) Chemical Synthesis

The invention provides hTRT polypeptides synthesized, entirely or inpart, using general chemical methods well known in the art (see e.g.,Caruthers et al., 1980, Nucleic Acids Res. Symp. Ser., 215-223; and Hornet al., 1980, Nucleic Acids Res. Symp. Ser., 225-232). For example,peptide synthesis can be performed using various solid-phase techniques(Roberge, et al., 1995, Science 269:202), including automated synthesis(e.g., using the Perkin Elmer ABI 431A Peptide Synthesizer in accordancewith the instructions provided by the manufacturer). When full lengthprotein is desired, shorter polypeptides may be fused by condensation ofthe amino terminus of one molecule with the carboxyl terminus of theother molecule to form a peptide bond.

The newly synthesized peptide can be substantially purified, forexample, by preparative high performance liquid chromatography (e.g.,Creighton, Proteins, Structures and Molecular Principles, WH Freeman andCo, New York N.Y. [1983]). The composition of the synthetic peptides (orany other peptides or polypeptides of the invention) may be confirmed byamino acid analysis or sequencing (e.g., the Edman degradationprocedure; Creighton, supra). Importantly, the amino acid sequence ofhTRT, or any part thereof, may be altered during direct synthesis and/orcombined using chemical methods with sequences from other proteins orotherwise, or any part thereof or for any purpose, to produce a variantpolypeptide of the invention.

2) Recombinant Expression of hTRT and Other TRT Proteins

The present invention provides methods, reagents, vectors, and cellsuseful for expression of hTRT polypeptides and nucleic acids using invitro (cell-free), ex vivo or in vivo (cell or organism-based)recombinant expression systems. In one embodiment, expression of thehTRT protein, or fragment thereof, comprises inserting the codingsequence into an appropriate expression vector (i.e., a vector thatcontains the necessary elements for the transcription and translation ofthe inserted coding sequence required for the expression systememployed). Thus, in one aspect, the invention provides for apolynucleotide substantially identical in sequence to an hTRT genecoding sequence at least 25 nucleotides, and preferably for manyapplications 50 to 100 nucleotides or more, of the hTRT cDNAs or genesof the invention, which is operably linked to a promoter to form atranscription unit capable of expressing an hTRT polypeptide. Methodswell known to those skilled in the art can be used to construct theexpression vectors containing an hTRT sequence and appropriatetranscriptional or translational controls provided by the presentinvention (see, e.g., Sambrook et al., supra, Ausubel et al. supra, andthis disclosure).

The hTRT polypeptides provided by the invention include fusion proteinsthat contain hTRT polypeptides or fragments of the hTRT protein. Thefusion proteins are typically produced by recombinant means, althoughthey may also be made by chemical synthesis. Fusion proteins can beuseful in providing enhanced expression of the hTRT polypeptideconstructs, or in producing hTRT polypeptides having other desirableproperties, for example, comprising a label (such as an enzymaticreporter group), binding group, or antibody epitope. An exemplary fusionprotein, comprising hTRT and enhanced green fluorescent protein (EGFP)sequences is described in Example 15, infra. It will be apparent to oneof skill that the uses and applications discussed in Example 15 andelsewhere herein are not limited to the particular fusion protein, butare illustrative of the uses of various fusion constructs.

The fusion protein systems of the invention can also be used tofacilitate efficient production and isolation of hTRT proteins orpeptides. For example, in some embodiments, the non-hTRT sequenceportion of the fusion protein comprises a short peptide that can bespecifically bound to an immobilized molecule such that the fusionprotein can be separated from unbound components (such as unrelatedproteins in a cell lysate). One example is a peptide sequence that isbound by a specific antibody. Another example is a peptide comprisingpolyhistidine tracts e.g. (His)₆ or histidine-tryptophan sequences thatcan be bound by a resin containing nickel or copper ions (i.e.,metal-chelate affinity chromatography). Other examples include Protein Adomains or fragments, which allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS, extension/affinitypurification system (Immunex Corp, Seattle Wash.). In some embodiments,the fusion protein includes a cleavage site so that the hTRT or otherTRT polypeptide sequence can be easily separated from the non-hTRTpeptide or protein sequence. In this case, cleavage may be chemical(e.g., cyanogen bromide,2-(2-nitrophenylsulphenyl)-3-methyl-3′-bromoindolene, hydroxylamine, orlow pH) or enzymatic (e.g., Factor Xa, enterokinase). The choice of thefusion and cleavage systems may depend, in part, on the portion (i.e.,sequence) of the hTRT polypeptide being expressed. Fusion proteinsgenerally are described in Ausubel et al., supra, Ch. 16, Kroll et al.,1993, DNA Cell. Biol. 12:441, and the Invitrogen 1997 Catalog(Invitrogen Inc, San Diego Calif.). Other exemplary fusion proteins ofthe invention with epitope tags or tags and cleavage sites are providedin Example 6, infra.

It will be appreciated by those of skill that, although the expressionsystems discussed in this section are focused on expression of hTRTpolypeptides, the same or similar cells, vectors and methods may be usedto express hTRT polynucleotides of the invention, including sense andantisense polynucleotides without necessarily desiring production ofhTRT polypeptides. Typically, expression of a polypeptide requires asuitable initiation codon (e.g., methionine), open reading frame, andtranslational regulatory signals (e.g., a ribosome binding site, atermination codon) which may be omitted when translation of a nucleicacid sequence to produce a protein is not desired.

Expression of hTRT polypeptides and polynucleotides may be carried outto accomplish any of several related benefits provided by the presentinvention. One illustrative benefit is expression of hTRT polypeptidesthat are subsequently isolated from the cell in which they are expressed(for example for production of large amounts of hTRT for use as avaccine or in screening applications to identify compounds that modulatetelomerase activity). A second illustrative benefit is expression ofhTRT in a cell to change the phenotype of the cell (as in gene therapyapplications). Nonmammalian cells can be used for expression of hTRT forpurification, while eukaryotic especially mammalian cells (e.g., humancells) can be used not only for isolation and purification of hTRT butalso for expression of hTRT when a change in phenotype in a cell isdesired (e.g., to effect a change in proliferative capacity as in genetherapy applications). By way of illustration and not limitation, hTRTpolypeptides having one or more telomerase activities (e.g. telomerasecatalytic activity) can be expressed in a host cell to increase theproliferative capacity of a cell (e.g., immortalize a cell) and,conversely, hTRT antisense polynucleotides or inhibitory polypeptidestypically can be expressed to reduce the proliferative capacity of acell (e.g., of a telomerase positive malignant tumor cell). Numerousspecific applications are described herein, e.g., in the discussion ofuses of the reagents and methods of the invention for therapeuticapplications, below.

Illustrative useful expression systems (cells, regulatory elements,vectors and expression) of the present invention include a number ofcell-free systems such as reticulocyte lysate and wheat germ systemsusing hTRT polynucleotides in accordance with general methods well knownin the art (see, e.g., Ausubel et al. supra at Ch. 10). In alternativeembodiments, the invention provides reagents and methods for expressinghTRT in prokaryotic or eukaryotic cells. Thus, the present inventionprovides nucleic acids encoding hTRT polynucleotides, proteins, proteinsubsequences, or fusion proteins that can be expressed in bacteria,fungi, plant, insect, and animal, including human cell expressionsystems known in the art, including isolated cells, cell lines, cellcultures, tissues, and whole organisms. As will be understood by thoseof skill, the hTRT polynucleotides introduced into a host cell or cellfree expression system will usually be operably linked to appropriateexpression control sequences for each host or cell free system.

Useful bacterial expression systems include E. coli, bacilli (such asBacillus subtilus), other enterobacteriaceae (such as Salmonella,Serratia, and various Pseudomonas species) or other bacterial hosts(e.g., Streptococcus cremoris, Streptococcus lactis, Streptococcusthermophilus, Leuconostoc citrovorum, Leuconostoc mesenteroides,Lactobacillus acidophilus, Lactobacillus lactis, Bifidobacteriumbifidum, Bifidobacteriu breve, and Bifidobacterium longum). The hTRTexpression constructs useful in prokaryotes include recombinantbacteriophage, plasmid or cosmid DNA expression vectors, or the like,and typically include promoter sequences. Illustrative promoters includeinducible promoters, such as the lac promoter, the hybrid lacZ promoterof the Bluescript7 phagemid [Stratagene, La Jolla Calif.] or pSport1[Gibco BRL]; phage lambda promoter systems; a tryptophan (trp) promotersystem; and ptrp-lac hybrids and the like. Bacterial expressionconstructs optionally include a ribosome binding site and transcriptiontermination signal regulatory sequences. Illustrative examples ofspecific vectors useful for expression include, for example, pTrcHis2,(Invitrogen, San Diego Calif.), pThioHis A, B & C, and numerous othersknown in the art or that may be developed (see, e.g. Ausubel). Usefulvectors for bacteria include those that facilitate production ofhTRT-fusion proteins. Useful vectors for high level expression of fusionproteins in bacterial cells include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBluescript7 (Stratagene), noted above, in which the sequence encodinghTRT protein, an hTRT fusion protein or an hTRT fragment may be ligatedinto the vector in-frame with sequences for the amino-terminal Met andthe subsequent 7 residues of β-galactosidase so that a hybrid protein isproduced (e.g., pIN vectors; Van Heeke and Schuster, 1989, J. Biol.Chem., 264:5503). Vectors such as pGEX vectors (e.g., pGEX-2TK;Pharmacia Biotech) may also be used to express foreign polypeptides,such as hTRT protein, as fusion proteins with glutathione S-transferase(GST). Such fusion proteins may be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems ofteninclude enterokinase, thrombin or factor Xa protease cleavage sites sothat the cloned polypeptide of interest can be released from the GSTmoiety at will, as may be useful in purification or other applications.Other examples are fusion proteins comprising hTRT and the E. coliMaltose Binding Protein (MBP) or E. Coli thioredoxin. Illustrativeexamples of hTRT expression constructs useful in bacterial cells areprovided in Example 6, infra.

The invention further provides hTRT polypeptides expressed in fungalsystems, such as Dictyostelium and, preferably, yeast, such asSaccharomyces cerevisiae, Pichia pastoris, Torulopsis holmil,Saccharomyces fragilis, Saccharomyces lactis, Hansenula polymorpha andCandida pseudotropicalis. When hTRT is expressed in yeast, a number ofsuitable vectors are available, including plasmid and yeast artificialchromosomes (YACs) vectors. The vectors typically include expressioncontrol sequences, such as constitutive or inducible promoters (e.g.,such as alpha factor, alcohol oxidase, PGH, and 3-phosphoglyceratekinase or other glycolytic enzymes), and an origin of replication,termination sequences and the like, as desired. Suitable vectors for usein Pichia include pPICZ, His6/pPICZB, pPICZalpha, pPIC3.5K, pPIC9K,pA0815, pGAP2A, B & C, pGAP2alpha A, B, and C (Invitrogen, San Diego,Calif.) and numerous others known in the art or to be developed. In oneembodiment, the vector His6/pPICZB (Invitrogen, San Diego, Calif.) isused to express a His₆-hTRT fusion protein in the yeast Pichia pastoris.An example of a vector useful in Saccharomyces is pYES2 (Invitrogen, SanDiego, Calif.). Illustrative examples of hTRT expression constructsuseful in yeast are provided in Example 6, infra.

The hTRT polypeptides of the invention may also be expressed in plantcell systems transfected with plant or plant virus expression vectors(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with bacterial expression vectors (e.g., Ti or pBR₃₂₂plasmid). In cases where plant virus expression vectors are used, theexpression of an hTRT-encoding sequence may be driven by any of a numberof promoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV (Brisson et al., 1984, Nature 310:511-514) may be usedalone or in combination with the omega leader sequence from TMV(Takamatsu et al., 1987, EMBO J., 6:307-311). Alternatively, plantpromoters such as that from the small subunit gene of RUBISCO (Coruzziet al., 1984, EMBO J., 3:1671-1680; Broglie et al., 1984, Science224:838-843) or heat shock promoters (Winter and Sinibaldi, 1991,Results Probl. Cell Differ., 17:85), or storage protein gene promotersmay be used. These constructs can be introduced into plant cells bydirect DNA transformation or pathogen-mediated transfection (for reviewsof such techniques, see Hobbs or Murry, 1992, in McGraw Hill Yearbook ofScience and Technology McGraw Hill New York N.Y., pp. 191-196 [1992]; orWeissbach and Weissbach, 1988, Methods for Plant Molecular Biology,Academic Press, New York N.Y., pp. 421-463).

Another expression system provided by the invention for expression ofhTRT protein is an insect system. A preferred system uses a baculoviruspolyhedrin promoter. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequenceencoding the gene of interest may be cloned into a nonessential regionof the virus, such as the polyhedrin gene, and placed under control ofthe polyhedrin promoter. Successful insertion of the sequence, e.g.,encoding the hTRT protein, will render the polyhedrin gene inactive andproduce recombinant virus lacking coat protein. The recombinant virusesare then used to infect S. frugiperda cells or Trichoplusia larvae, inwhich the hTRT sequence is then expressed (see, for general methods,Smith et al., J. Virol., 46:584 [1983]; Engelhard et al., Proc. Natl.Acad. Sci. 91:3224-7 [1994]). Useful vectors for baculovirus expressioninclude pBlueBacHis2 A, B & C, pBlueBac4.5, pMelBacB and numerous othersknown in the art or to be developed. Illustrative examples of hTRTexpression constructs useful in insect cells are provided in Example 6,infra.

The present invention also provides expression systems in mammals andmammalian cells. As noted supra, hTRT polynucleotides may be expressedin mammalian cells (e.g., human cells) for production of significantquantities of hTRT polypeptides (e.g., for purification) or to changethe phenotype of a target cell (e.g., for purposes of gene therapy, cellimmortalization, or other). In the latter case, the hTRT polynucleotideexpressed may or may not encode a polypeptide with a telomerasecatalytic activity. That is, expression may be of a sense or antisensepolynucleotide, an inhibitory or stimulatory polypeptide, a polypeptidewith zero, one or more telomerase activities, and other combinations andvariants disclosed herein or apparent to one of skill upon review ofthis disclosure.

Suitable mammalian host tissue culture cells for expressing the nucleicacids of the invention include any normal mortal or normal or abnormalimmortal animal or human cell, including: monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293; Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidneycells (BHK, ATCC CCL 10); CHO (ATCC CCL 61 and CRL 9618); mouse sertolicells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidneycells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76,ATCC CRL 1587); human cervical carcinoma cells (HeLa, ATCC CCL 2);canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human livercells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);TRI cells (Mather, et al., Annals N.Y. Acad. Sci. 383:44-46 (1982); MDCKcells (ATCC CCL 34 and CRL 6253); HEK 293 cells (ATCC CRL 1573); andWI-38 cells (ATCC CCL 75; ATCC: American Type Culture Collection,Rockville, Md.). The use of mammalian tissue cell culture to expresspolypeptides is discussed generally in Winnacker, From Genes to Clones(VCH Publishers, N.Y., N.Y., 1987).

For mammalian host cells, viral-based and nonviral expression systemsare provided. Nonviral vectors and systems include plasmids and episomalvectors, typically with an expression cassette for expressing a proteinor RNA, and human artificial chromosomes (see, e.g., Harrington et al.,1997, Nat Genet 15:345). For example, nonviral vectors useful forexpression of hTRT polynucleotides and polypeptides in mammalian (e.g.,human) cells include pcDNA3.1/His, pEBVHis A, B & C, (Invitrogen, SanDiego Calif.), MPSV vectors, others described in the Invitrogen 1997Catalog (Invitrogen Inc, San Diego Calif.), which is incorporated in itsentirety herein, and numerous others known in the art for otherproteins. Illustrative examples of hTRT expression constructs useful inmammalian cells are provided in Example 6, infra.

Useful viral vectors include vectors based on retroviruses,adenoviruses, adenoassociated viruses, herpes viruses, vectors based onSV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectorsand Semliki Forest virus (SFV). SFV and vaccinia vectors are discussedgenerally in Ausubel et al., supra, Ch 16. These vectors are often madeup of two components, a modified viral genome and a coat structuresurrounding it (see generally Smith, 1995, Annu. Rev. Microbiol. 49:807), although sometimes viral vectors are introduced in naked form orcoated with proteins other than viral proteins. However, the viralnucleic acid in a vector may be changed in many ways, for example, whendesigned for gene therapy. The goals of these changes are to disablegrowth of the virus in target cells while maintaining its ability togrow in vector form in available packaging or helper cells, to providespace within the viral genome for insertion of exogenous DNA sequences,and to incorporate new sequences that encode and enable appropriateexpression of the gene of interest. Thus, vector nucleic acids generallycomprise two components: essential cis-acting viral sequences forreplication and packaging in a helper line and the transcription unitfor the exogenous gene. Other viral functions are expressed in trans ina specific packaging or helper cell line. Adenoviral vectors (e.g., foruse in human gene therapy) are described in, e.g., Rosenfeld et al.,1992, Cell 68: 143; PCT publications WO 94/12650; 94/12649; and94/12629. In cases where an adenovirus is used as an expression vector,a sequence encoding hTRT may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a nonessential E1 or E3 regionof the viral genome will result in a viable virus capable of expressingin infected host cells (Logan and Shenk, 1984, Proc. Natl. Acad. Sci.,81:3655). Replication-defective retroviral vectors harboring atherapeutic polynucleotide sequence as part of the retroviral genome aredescribed in, e.g., Miller et al., 1990, Mol. Cell. Biol. 10: 4239;Kolberg, 1992, J. NIH Res. 4: 43; and Cornetta et al., 1991, Hum. GeneTher. 2: 215.

In mammalian cell systems, promoters from mammalian genes or frommammalian viruses are often appropriate. Suitable promoters may beconstitutive, cell type-specific, stage-specific, and/or modulatable orregulatable (e.g., by hormones such as glucocorticoids). Usefulpromoters include, but are not limited to, the metallothionein promoter,the constitutive adenovirus major late promoter, thedexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIIIpromoter, the constitutive MPSV promoter, the tetracycline-inducible CMVpromoter (such as the human immediate-early CMV promoter), theconstitutive CMV promoter, and promoter-enhancer combinations known inthe art.

Other regulatory elements may also be required or desired for efficientexpression of an hTRT polynucleotide and/or translation of a sequenceencoding hTRT proteins. For translation, these elements typicallyinclude an ATG initiation codon and adjacent ribosome binding site orother sequences. For sequences encoding the hTRT protein, provided itsinitiation codon and upstream promoter sequences are inserted into anexpression vector, no additional translational or other control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous transcriptional and/ortranslational control signals (e.g., the promoter, ribosome-bindingsite, and ATG initiation codon) must often be provided. Furthermore, theinitiation codon must typically be in the correct reading frame toensure translation of the desired protein. Exogenous transcriptionalelements and initiation codons can be of various origins, both naturaland synthetic. In addition, the efficiency of expression may be enhancedby the inclusion of enhancers appropriate to the cell system in use(Scharf et al., 1994, Results Probl. Cell Differ. 20:125; and Bittner etal. 1987, Meth. Enzymol., 153:516). For example, the SV40 enhancer orCMV enhancer may be used to increase expression in mammalian host cells.

Expression of hTRT gene products can also by effected (increased) byactivation of an hTRT promoter or enhancer in a cell such as a humancell, e.g., a telomerase-negative cell line. Activation can be carriedout in a variety of ways, including administration of an exogenouspromoter activating agent, or inhibition of a cellular component thatsuppresses expression of the hTRT gene. It will be appreciated that,conversely, inhibition of promoter function, as described infra, willreduce hTRT gene expression.

The invention provides inducible and repressible expression of hTRTpolypeptides using such system as the Ecdysone-Inducible ExpressionSystem (Invitrogen), and the Tet-On and Tet-off tetracycline regulatedsystems from Clontech. The ecdysone-inducible expression system uses thesteroid hormone ecdysone analog, muristerone A, to activate expressionof a recombinant protein via a heterodimeric nuclear receptor (No etal., 1996, Proc. Natl. Acad. Sci. USA 93:3346). In one embodiment of theinvention, hTRT is cloned in the pIND vector (Clontech), which containsfive modified ecdysone response elements (E/GREs) upstream of a minimalheat shock promoter and the multiple cloning site. The construct is thentransfected in cell lines stably expressing the ecdysone receptor. Aftertransfection, cells are treated with muristerone A to induceintracellular expression from pIND. In another embodiment of theinvention, hTRT polypeptide is expressed using the Tet-on and Tet-offexpression systems (Clontech) to provide regulated, high-level geneexpression (Gossen et al., 1992, Proc. Natl. Acad. Sci. USA 89:5547;Gossen et al., 1995, Science 268:1766).

The hTRT vectors of the invention may be introduced into a cell, tissue,organ, patient or animal by a variety of methods. The nucleic acidexpression vectors (typically dsDNA) of the invention can be transferredinto the chosen host cell by well-known methods such as calcium chloridetransformation (for bacterial systems), electroporation, calciumphosphate treatment, liposome-mediated transformation, injection andmicroinjection, ballistic methods, virosomes, immunoliposomes,polycation:nucleic acid conjugates, naked DNA, artificial virions,fusion to the herpes virus structural protein VP22 (Elliot and O'Hare,Cell 88:223), agent-enhanced uptake of DNA, and ex vivo transduction.Useful liposome-mediated DNA transfer methods are described in U.S. Pat.Nos. 5,049,386, 4,946,787; and 4,897,355; PCT publications WO 91/17424,WO 91/16024; Wang and Huang, 1987, Biochem. Biophys. Res. Commun. 147:980; Wang and Huang, 1989, Biochemistry 28: 9508; Litzinger and Huang,1992, Biochem. Biophys. Acta 1113:201; Gao and Huang, 1991, Biochem.Biophys. Res. Commun. 179: 280. Immunoliposomes have been described ascarriers of exogenous polynucleotides (Wang and Huang, 1987, Proc. Natl.Acad. Sci. U.S.A. 84:7851; Trubetskoy et al., 1992, Biochem. Biophys.Acta 1131:311) and may have improved cell type specificity as comparedto liposomes by virtue of the inclusion of specific antibodies whichpresumably bind to surface antigens on specific cell types. Behr et al.,1989, Proc. Natl. Acad. Sci. U.S.A. 86:6982 report using lipopolyamineas a reagent to mediate transfection itself, without the necessity ofany additional phospholipid to form liposomes. Suitable delivery methodswill be selected by practitioners in view of acceptable practices andregulatory requirements (e.g., for gene therapy or production of celllines for expression of recombinant proteins). It will be appreciatedthat the delivery methods listed above may be used for transfer ofnucleic acids into cells for purposes of gene therapy, transfer intotissue culture cells, and the like.

For long-term, high-yield production of recombinant proteins, stableexpression will often be desired. For example, cell lines which stablyexpress hTRT can be prepared using expression vectors of the inventionwhich contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells may be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth of cells which successfully express the introducedsequences in selective media. Resistant, stably transfected cells can beproliferated using tissue culture techniques appropriate to the celltype. An amplification step, e.g., by administration of methyltrexate tocells transfected with a DHFR gene according to methods well known inthe art, can be included.

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,phosphorylation, lipidation and acylation. Post-translational processingmay also be important for correct insertion, folding and/or function.Different host cells have cellular machinery and characteristicmechanisms specific for each cell for such post-translational activitiesand so a particular cell may be chosen to ensure the correctmodification and processing of the introduced, foreign protein.

As noted supra, when expressing an hTRT protein (including variants) incells or organisms it is sometimes desirable to use an hTRTprotein-encoding polynucleotide that employs a codon distribution otherthan that found in a naturally occurring hTRT gene. hTRTprotein-encoding polynucleotides with alternative codons throughout, orat specific sites, in the coding sequence are used to optimize (e.g.,increase) expression of the hTRT protein in cells, especially non-humancells (e.g., bacterial, plant, fungal, and non-human animal cells) whichhave different preferential codon usage than human cells. Codon changesmay also be used to facilitate manipulation of the hTRT polynucleotide(e.g., by engineering useful tags or restriction sites into the codingsequence), and for other reasons. When the goal is to optimizeexpression (e.g., by increasing translational efficiency), tables ofpreferred codon usage, which are publicly available and are well knownto those of skill, are used to design a suitable polynucleotide by“reverse translation” of the desired (e.g., hTRT) amino acid sequence.Alternatively, preferred codon usage can be determined for a particularorganism (e.g., Pichia pastoris) or class of genes (e.g., highlyexpressed genes of a particular organism) by comparison of publishedgene sequences for the target organism or gene class.

Illustrative hTRT-encoding polynucleotide sequences are provided inTable 9 (A-E), infra. All of the sequences in Table 9 are in the 5′→3′.Table 9A shows an hTRT protein encoding polynucleotide that uses a codondistribution preferentially employed in the bacterium E. coli. Table 9Bshows a second polynucleotide sequence particularly useful forexpression in E. coli (and other enteric bacteria) using codonspreferentially used in highly expressed genes in enteric bacteria. Table4C shows an hTRT protein encoding polynucleotide that uses a codondistribution preferentially employed in yeast (i.e., S. cerevisiae).Table 4D shows an hTRT protein encoding polynucleotide that uses a codondistribution preferentially used in highly expressed genes in yeast.Table 4E shows an hTRT protein encoding polynucleotide that uses a“generic” codon distribution that should be efficiently expressed inboth bacteria (e.g., E. coli) and yeast (e.g., S. pombe, S. cerevisiae,P. pastoris) and some insect (e.g., S. frugiperda) cells. Such “generic”polynucleotide sequences (optimized for more than one organism) areuseful for, for example, comparative studies, screening in differentorganisms of hTRT binding or modulatory agents, creation of shuttlevectors, and other uses. In this “generic” sequence, the codon TCT(serine) may not be optimal for expression in Drosophila cells.Therefore, in an alternative embodiment the sequence in Table 4E ismodified to replace TCT with TCC for efficient expression in Drosophilaas well as bacteria and yeast.

TABLE 9 hTRT-ENCODING POLYNUCLEOTIDE SEQUENCES EMPLOYING ALTERNATIVECODON DISTRIBUTIONS TABLE 9A E. coli (all genes) (SEQ ID NO:638) ATG CCGCGC GCG CCG CGC TGC CGC GCG GTG CGC AGC CTG CTG CGC AGC CAT TAT CGC GAAGTG CTG CCG CTG GCG ACC TTT GTG CGC CGC CTG GGC CCG CAG GGC TGG CGd CTGGTG CAG CGC GGC GAT CCG GCG GCG TTT CGC GCG CTG GTG GCG CAG TGC CTG GTGTGC GTG CCG TGG GAT GCG CGC CCG CCG CCG GCG GCG CCG AGC TTT CGC CAG GTGAGC TGC CTG AAA GAA CTG GTG GCG CGC GTG CTG CAG CGC CTG TGC GAA CGC GGCGCG AAA AAC GTG CTG GCG TTT GGC TTT GCG CTG CTG GAT GGC GCG CGC GGC GGCCCG CCG GAA GCG TTT ACC ACC AGC GTG CGC AGC TAT CTG CCG AAC ACC GTG ACCGAT GCG CTG CGC GGC AGC GGC GCG TGG GGC CTG CTG CTG CGC CGC GTG GGC GATGAT GTG CTG GTG CAT CTG CTG GCG CGC TGC GCG CTG TTT GTG CTG GTG GCG CCGAGC TGC GCG TAT CAG GTG TGC GGC CCG CCG CTG TAT CAG CTG GGC GCG GCG ACCCAG GCG CGC CCG CCG CCG CAT GCG AGC GGC CCG CGC CGC CGC CTG GGC TGC GAACGC GCG TGG AAC CAT AGC GTG CGC GAA GCG GGC GTG CCG CTG GGC CTG CCG GCGCCG GGC GCG CGC CGC CGC GGC GGC AGC GCG AGC CGC AGC CTG CCG CTG CCG AAACGC CCG CGC CGC GGC GCG GCG CCG GAA CCG GAA CGC ACC CCG GTG GGC CAG GGCAGC TGG GCG CAT CCG GGC CGC ACC CGC GGC CCG AGC GAT CGC GGC TTT TGC GTGGTG AGC CCG GCG CGC CCG GCG GAA GAA GCG ACC AGC CTG GAA GGC GCG CTG AGCGGC ACC CGC CAT AGC CAT CCG AGC GTG GGC CGC CAG CAT CAT GCG GGC CCG CCGAGC ACC AGC CGC CCG CCG CGC CCG TGG GAT ACC CCG TGC CCG CCG GTG TAT GCGGAA ACC AAA CAT TTT CTG TAT AGC AGC GGC GAT AAA GAA CAG CTG CGC CCG AGCTTT CTG CTG AGC AGC CTG CGC CCG AGC CTG ACC GGC GCG CGC CGC CTG GTG GAAACC ATT TTT CTG GGC AGC CGC CCG TGG ATG CCG GGC ACC CCG CGC CGC CTG CCGCGC CTG CCG CAG CGC TAT TGG CAG ATG CGC CCG CTG TTT CTG GAA CTG CTG GGCAAC CAT GCG CAG TGC CCG TAT GGC GTG CTG CTG AAA ACC CAT TGC CCG CTG CGCGCG GCG GTG ACC CCG GCG GCG GGC GTG TGC GCG CGC GAA AAA CCG CAG GGC AGCGTG GCG GCG CCG GAA GAA GAA GAT ACC GAT CCG CGC CGC CTG GTG CAG CTG CTGCGC CAG CAT AGC AGC CCG TGG CAG GTG TAT GGC TTT GTG CGC GCG TGC CTG CGCCGC CTG GTG CCG CCG GGC CTG TGG GGC AGC CGC CAT AAC GAA CGC CGC TTT CTGCGC AAC ACC AAA AAA TTT ATT AGC CTG GGC AAA CAT GCG AAA CTG AGC CTG CAGGAA CTG ACC TGG AAA ATG AGC GTG CGC GAT TGC GCG TGG CTG CGC CGC AGC CCGGGC GTG GGC TGC GTG CCG GCG GCG GAA CAT CGC CTG CGC GAA GAA ATT CTG GCGAAA TTT CTG CAT TGG CTG ATG AGC GTG TAT GTG GTG GAA CTG CTG CGC AGC TTTTTT TAT GTG ACC GAA ACC ACC TTT CAG AAA AAC CGC CTG TTT TTT TAT CGC AAAAGC GTG TGG AGC AAA CTG CAG AGC ATT GGC ATT CGC CAG CAT CTG AAA CGC GTGCAG CTG CGC GAA CTG AGC GAA GCG GAA GTG CGC CAG CAT CGC GAA GCG CGC CCGGCG CTG CTG ACC AGC CGC CTG CGC TTT ATT CCG AAA CCG GAT GGC CTG CGC CCGATT GTG AAC ATG GAT TAT GTG GTG GGC GCG CGC ACC TTT CGC CGC GAA AAA CGCGCG GAA CGC CTG ACC AGC CGC GTG AAA GCG CTG TTT AGC GTG CTG AAC TAT GAACGC GCG CGC CGC CCG GGC CTG CTG GGC GCG AGC GTG CTG GGC CTG GAT GAT ATTCAT CGC GCG TGG CGC ACC TTT GTG CTG CGC GTG CGC GCG CAG GAT CCG CCG CCGGAA CTG TAT TTT GTG AAA GTG GAT GTG ACC GGC GCG TAT GAT ACC ATT CCG CAGGAT CGC CTG ACC GAA GTG ATT GCG AGC ATT ATT AAA CCG CAG AAC ACC TAT TGCGTG CGC CGC TAT GCG GTG GTG CAG AAA GCG GCG CAT GGC CAT GTG CGC AAA GCGTTT AAA AGC CAT GTG AGC ACC CTG ACC GAT CTG CAG CCG TAT ATG CGC CAG TTTGTG GCG CAT CTG CAG GAA ACC AGC CCG CTG CGC GAT GCG GTG GTG ATT GAA CAGAGC AGC AGC CTG AAC GAA GCG AGC AGC GGC CTG TTT GAT GTG TTT CTG CGC TTTATG TGC CAT CAT GCG GTG CGC ATT CGC GGC AAA AGC TAT GTG CAG TGC CAG GGCATT CCG CAG GGC AGC ATT CTG AGC ACC CTG CTG TGC AGC CTG TGC TAT GGC GATATG GAA AAC AAA CTG TTT GCG GGC ATT CGC CGC GAT GGC CTG CTG CTG CGC CTGGTG GAT GAT TTT CTG CTG GTG ACC CCG CAT CTG ACC CAT GCG AAA ACC TTT CTGCGC ACC CTG GTG CGC GGC GTG CCG GAA TAT GGC TGC GTG GTG AAC CTG CGC AAAACC GTG GTG AAC TTT CCG GTG GAA GAT GAA GCG CTG GGC GGC ACC GCG TTT GTGCAG ATG CCG GCG CAT GGC CTG TTT CCG TGG TGC GGC CTG CTG CTG GAT ACC CGCACC CTG GAA GTG CAG AGC GAT TAT AGC AGC TAT GCG CGC ACC AGC ATT CGC GCGAGC CTG ACC TTT AAC CGC GGC TTT AAA GCG GGC CGC AAC ATG CGC CGC AAA CTGTTT GGC GTG CTG CGC CTG AAA TGC CAT AGC CTG TTT CTG GAT CTG CAG GTG AACAGC CTG CAG ACC GTG TGC ACC AAC ATT TAT AAA ATT CTG CTG CTG CAG GCG TATCGC TTT CAT GCG TGC GTG CTG CAG CTG CCG TTT CAT CAG CAG GTG TGG AAA AACCCG ACC TTT TTT CTG CGC GTG ATT AGC GAT ACC GCG AGC CTG TGC TAT AGC ATTCTG AAA GCG AAA AAC GCG GGC ATG AGC CTG GGC GCG AAA GGC GCG GCG GGC CCGCTG CCG AGC GAA GCG GTG CAG TGG CTG TGC CAT CAG GCG TTT CTG CTG AAA CTGACC CGC CAT CGC GTG ACC TAT GTG CCG CTG CTG GGC AGC CTG CGC ACC GCG CAGACC CAG CTG AGC CGC AAA CTG CCG GGC ACC ACC CTG ACC GCG CTG GAA GCG GCGGCG AAC CCG GCG CTG CCG AGC GAT TTT AAA ACC ATT CTG GAT TABLE 9B EntericBacteria (High Expressing Genes) (SEQ ID NO:639)    1 ATGCCGCGTGCTCCGCGTTG CCGTGCTGTT CGTTCCCTGC TGCGTTCCCA   51 CTACCGTGAA GTTCTGCCGCTGGCTACCTT CGTTCGTCGT CTGGGTCCGC  101 AGGGTTGGCG TCTGGTTCAG CGTGGTGACCCGGCTGCTTT CCGTGCTCTG  151 GTTGCTCAGT GCCTGGTTTG CGTTCCGTGG GACGCTCGTCCGCCGCCGGC  201 TGCTCCGTCC TTCCGTCAGG TTTCCTGCCT GAAAGAACTG GTTGCTCGTG 251 TTCTGCAGCG TCTGTGCGAA CGTGGTGCTA AAAACGTTCT GGCTTTCGGT  301TTCGCTCTGC TGGACGGTGC TCGTGGTGGT CCGCCGGAAG CTTTCACCAC  351 CTCCGTTCGTTCCTACCTGC CGAACACCGT TACCGACGCT CTGCGTGGTT  401 CCGGTGCTTG GGGTCTGCTGCTGCGTCGTG TTGGTGACGA CGTTCTGGTT  451 CACCTGCTGG CTCGTTGCGC TCTGTTCGTTCTGGTTGCTC CGTCCTGCGC  501 TTACCAGGTT TGCGGTCCGC CGCTGTACCA GCTGGGTGCTGCTACCCAGG  551 CTCGTCCGCC GCCGCACGCT TCCGGTCCGC GTCGTCGTCT GGGTTGCGAA 601 CGTGCTTGGA ACCACTCCGT TCGTGAAGCT GGTGTTCCGC TGGGTCTGCC  651GGCTCCGGGT GCTCGTCGTC GTGGTGGTTC CGCTTCCCGT TCCCTGCCGC  701 TGCCGAAACGTCCGCGTCGT GGTGCTGCTC CGGAACCGGA ACGTACCCCG  751 GTTGGTCAGG GTTCCTGGGCTCACCCGGGT CGTACCCGTG GTCCGTCCGA  801 CCGTGGTTTC TGCGTTGTTT CCCCGGCTCGTCCGGCTGAA GAAGCTACCT  851 CCCTGGAAGG TGCTCTGTCC GGTACCCGTC ACTCCCACCCGTCCGTTGGT  901 CGTCAGCACC ACGCTGGTCC GCCGTCCACC TCCCGTCCGC CGCGTCCGTG 951 GGACACCCCG TGCCCGCCGG TTTACGCTGA AACCAAACAC TTCCTGTACT 1001CCTCCGGTGA CAAAGAACAG CTGCGTCCGT CCTTCCTGCT GTCCTCCCTG 1051 CGTCCGTCCCTGACCGGTGC TCGTCGTCTG GTTGAAACCA TCTTCCTGGG 1101 TTCCCGTCCG TGGATGCCGGGTACCCCGCG TCGTCTGCCG CGTCTGCCGC 1151 AGCGTTACTG GCAGATGCGT CCGCTGTTCCTGGAACTGCT GGGTAACCAC 1201 GCTCAGTGCC CGTACGGTGT TCTGCTGAAA ACCCACTGCCCGCTGCGTGC 1251 TGCTGTTACC CCGGCTGCTG GTGTTTGCGC TCGTGAAAAA CCGCAGGGTT1301 CCGTTGCTGC TCCGGAAGAA GAAGACACCG ACCCGCGTCG TCTGGTTCAG 1351CTGCTGCGTC AGCACTCCTC CCCGTGGCAG GTTTACGGTT TCGTTCGTGC 1401 TTGCCTGCGTCGTCTGGTTC CGCCGGGTCT GTGGGGTTCC CGTCACAACG 1451 AACGTCGTTT CCTGCGTAACACCAAAAAAT TCATCTCCCT GGGTAAACAC 1501 GCTAAACTGT CCCTGCAGGA ACTGACCTGGAAAATGTCCG TTCGTGACTG 1551 CGCTTGGCTG CGTCGTTCCC CGGGTGTTGG TTGCGTTCCGGCTGCTGAAC 1601 ACCGTCTGCG TGAAGAAATC CTGGCTAAAT TCCTGCACTG GCTGATGTCC1651 GTTTACGTTG TTGAACTGCT GCGTTCCTTC TTCTACGTTA CCGAAACCAC 1701CTTCCAGAAA AACCGTCTGT TCTTCTACCG TAAATCCGTT TGGTCCAAAC 1751 TGCAGTCCATCGGTATCCGT CAGCACCTGA AACGTGTTCA GCTGCGTGAA 1801 CTGTCCGAAG CTGAAGTTCGTCAGCACCGT GAAGCTCGTC CGGCTCTGCT 1851 GACCTCCCGT CTGCGTTTCA TCCCGAAACCGGACGGTCTG CGTCCGATCG 1901 TTAACATGGA CTACGTTGTT GGTGCTCGTA CCTTCCGTCGTGAAAAACGT 1951 GCTGAACGTC TGACCTCCCG TGTTAAAGCT CTGTTCTCCG TTCTGAACTA2001 CGAACGTGCT CGTCGTCCGG GTCTGCTGGG TGCTTCCGTT CTGGGTCTGG 2051ACGACATCCA CCGTGCTTGG CGTACCTTCG TTCTGCGTGT TCGTGCTCAG 2101 GACCCGCCGCCGGAACTGTA CTTCGTTAAA GTTGACGTTA CCGGTGCTTA 2151 CGACACCATC CCGCAGGACCGTCTGACCGA AGTTATCGCT TCCATCATCA 2201 AACCGCAGAA CACCTACTGC GTTCGTCGTTACGCTGTTGT TCAGAAAGCT 2251 GCTCACGGTC ACGTTCGTAA AGCTTTCAAA TCCCACGTTTCCACCCTGAC 2301 CGACCTGCAG CCGTACATGC GTCAGTTCGT TGCTCACCTG CAGGAAACCT2351 CCCCGCTGCG TGACGCTGTT GTTATCGAAC AGTCCTCCTC CCTGAACGAA 2401GCTTCCTCCG GTCTGTTCGA CGTTTTCCTG CGTTTCATGT GCCACCACGC 2451 TGTTCGTATCCGTGGTAAAT CCTACGTTCA GTGCCAGGGT ATCCCGCAGG 2501 GTTCCATCCT GTCCACCCTGCTGTGCTCCC TGTGCTACGG TGACATGGAA 2551 AACAAACTGT TCGCTGGTAT CCGTCGTGACGGTCTGCTGC TGCGTCTGGT 2601 TGACGACTTC CTGCTGGTTA CCCCGCACCT GACCCACGCTAAAACCTTCC 2651 TGCGTACCCT GGTTCGTGGT GTTCCGGAAT ACGGTTGCGT TGTTAACCTG2701 CGTAAAACCG TTGTTAACTT CCCGGTTGAA GACGAAGCTC TGGGTGGTAC 2751CGCTTTCGTT CAGATGCCGG CTCACGGTCT GTTCCCGTGG TGCGGTCTGC 2801 TGCTGGACACCCGTACCCTG GAAGTTCAGT CCGACTACTC CTCCTACGCT 2851 CGTACCTCCA TCCGTGCTTCCCTGACCTTC AACCGTGGTT TCAAAGCTGG 2901 TCGTAACATG CGTCGTAAAC TGTTCGGTGTTCTGCGTCTG AAATGCCACT 2951 CCCTGTTCCT GGACCTGCAG GTTAACTCCC TGCAGACCGTTTGCACCAAC 3001 ATCTACAAAA TCCTGCTGCT GCAGGCTTAC CGTTTCCACG CTTGCGTTCT3051 GCAGCTGCCG TTCCACCAGC AGGTTTGGAA AAACCCGACC TTCTTCCTGC 3101GTGTTATCTC CGACACCGCT TCCCTGTGCT ACTCCATCCT GAAAGCTAAA 3151 AACGCTGGTATGTCCCTGGG TGCTAAAGGT GCTGCTGGTC CGCTGCCGTC 3201 CGAAGCTGTT CAGTGGCTGTGCCACCAGGC TTTCCTGCTG AAACTGACCC 3251 GTCACCGTGT TACCTACGTT CCGCTGCTGGGTTCCCTGCG TACCGCTCAG 3301 ACCCAGCTGT CCCGTAAACT GCCGGGTACC ACCCTGACCGCTCTGGAAGC 3351 TGCTGCTAAC CCGGCTCTGC CGTCCGACTT CAAAACCATC CTGGAC    TABLE 9C Yeast (All Genes) (SEQ ID NO:640) ATG CCA AGA GCT CCA AGA TGTAGA GCT GTT AGA TCT TTG TTG AGA TCT CAT TAT AGA GAA GTT TTG CCA TTG GCTACT TTT GTT AGA AGA TTG GGT CCA CAA GGT TGG AGA TTG GTT CAA AGA GGT GATCCA GCT GCT TTT AGA GCT TTG GTT GCT CAA TGT TTG GTT TGT GTT CCA TGG GATGCT AGA CCA CCA CCA GCT GCT CCA TCT TTT AGA CAA GTT TCT TGT TTG AAA GAATTG GTT GCT AGA GTT TTG CAA AGA TTG TGT GAA AGA GGT GCT AAA AAT GTT TTGGCT TTT GGT TTT GCT TTG TTG GAT GGT GCT AGA GGT GGT CCA CCA GAA GCT TTTACT ACT TCT GTT AGA TCT TAT TTG CCA AAT ACT GTT ACT GAT GCT TTG AGA GGTTCT GGT GCT TGG GGT TTG TTG TTG AGA AGA GTT GGT GAT GAT GTT TTG GTT CATTTG TTG GCT AGA TGT GCT TTG TTT GTT TTG GTT GCT CCA TCT TGT GCT TAT CAAGTT TGT GGT CCA CCA TTG TAT CAA TTG GGT GCT GCT ACT CAA GCT AGA CCA CCACCA CAT GCT TCT GGT CCA AGA AGA AGA TTG GGT TGT GAA AGA GCT TGG AAT CATTCT GTT AGA GAA GCT GGT GTT CCA TTG GGT TTG CCA GCT CCA GGT GCT AGA AGAAGA GGT GGT TCT GCT TCT AGA TCT TTG CCA TTG CCA AAA AGA CCA AGA AGA GGTGCT GCT CCA GAA CCA GAA AGA ACT CCA GTT GGT CAA GGT TCT TGG GCT CAT CCAGGT AGA ACT AGA GGT CCA TCT GAT AGA GGT TTT TGT GTT GTT TCT CCA GCT AGACCA GCT GAA GAA GCT ACT TCT TTG GAA GGT GCT TTG TCT GGT ACT AGA CAT TCTCAT CCA TCT GTT GGT AGA CAA CAT CAT GCT GGT CCA CCA TCT ACT TCT AGA CCACCA AGA CCA TGG GAT ACT CCA TGT CCA CCA GTT TAT GCT GAA ACT AAA CAT TTTTTG TAT TCT TCT GGT GAT AAA GAA CAA TTG AGA CCA TCT TTT TTG TTG TCT TCTTTG AGA CCA TCT TTG ACT GGT GCT AGA AGA TTG GTT GAA ACT ATT TTT TTG GGTTCT AGA CCA TGG ATG CCA GGT ACT CCA AGA AGA TTG CCA AGA TTG CCA CAA AGATAT TGG CAA ATG AGA CCA TTG TTT TTG GAA TTG TTG GGT AAT CAT GCT CAA TGTCCA TAT GGT GTT TTG TTG AAA ACT CAT TGT CCA TTG AGA GCT GCT GTT ACT CCAGCT GCT GGT GTT TGT GCT AGA GAA AAA CCA CAA GGT TCT GTT GCT GCT CCA GAAGAA GAA GAT ACT GAT CCA AGA AGA TTG GTT CAA TTG TTG AGA CAA CAT TCT TCTCCA TGG CAA GTT TAT GGT TTT GTT AGA GCT TGT TTG AGA AGA TTG GTT CCA CCAGGT TTG TGG GGT TCT AGA CAT AAT GAA AGA AGA TTT TTG AGA AAT ACT AAA AAATTT ATT TCT TTG GGT AAA CAT GCT AAA TTG TCT TTG CAA GAA TTG ACT TGG AAAATG TCT GTT AGA GAT TGT GCT TGG TTG AGA AGA TCT CCA GGT GTT GGT TGT GTTCCA GCT GCT GAA CAT AGA TTG AGA GAA GAA ATT TTG GCT AAA TTT TTG CAT TGGTTG ATG TCT GTT TAT GTT GTT GAA TTG TTG AGA TCT TTT TTT TAT GTT ACT GAAACT ACT TTT CAA AAA AAT AGA TTG TTT TTT TAT AGA AAA TCT GTT TGG TCT AAATTG CAA TCT ATT GGT ATT AGA CAA CAT TTG AAA AGA GTT CAA TTG AGA GAA TTGTCT GAA GCT GAA GTT AGA CAA CAT AGA GAA GCT AGA CCA GCT TTG TTG ACT TCTAGA TTG AGA TTT ATT CCA AAA CCA GAT GGT TTG AGA CCA ATT GTT AAT ATG GATTAT GTT GTT GGT GCT AGA ACT TTT AGA AGA GAA AAA AGA GCT GAA AGA TTG ACTTCT AGA GTT AAA GCT TTG TTT TCT GTT TTG AAT TAT GAA AGA GCT AGA AGA CCAGGT TTG TTG GGT GCT TCT GTT TTG GGT TTG GAT GAT ATT CAT AGA GCT TGG AGAACT TTT GTT TTG AGA GTT AGA GCT CAA GAT CCA CCA CCA GAA TTG TAT TTT GTTAAA GTT GAT GTT ACT GGT GCT TAT GAT ACT ATT CCA CAA GAT AGA TTG ACT GAAGTT ATT GCT TCT ATT ATT AAA CCA CAA AAT ACT TAT TGT GTT AGA AGA TAT GCTGTT GTT CAA AAA GCT GCT CAT GGT CAT GTT AGA AAA GCT TTT AAA TCT CAT GTTTCT ACT TTG ACT GAT TTG CAA CCA TAT ATG AGA CAA TTT GTT GCT CAT TTG CAAGAA ACT TCT CCA TTG AGA GAT GCT GTT GTT ATT GAA CAA TCT TCT TCT TTG AATGAA GCT TCT TCT GGT TTG TTT GAT GTT TTT TTG AGA TTT ATG TGT CAT CAT GCTGTT AGA ATT AGA GGT AAA TCT TAT GTT CAA TGT CAA GGT ATT CCA CAA GGT TCTATT TTG TCT ACT TTG TTG TGT TCT TTG TGT TAT GGT GAT ATG GAA AAT AAA TTGTTT GCT GGT ATT AGA AGA GAT GGT TTG TTG TTG AGA TTG GTT GAT GAT TTT TTGTTG GTT ACT CCA CAT TTG ACT CAT GCT AAA ACT TTT TTG AGA ACT TTG GTT AGAGGT GTT CCA GAA TAT GGT TGT GTT GTT AAT TTG AGA AAA ACT GTT GTT AAT TTTCCA GTT GAA GAT GAA GCT TTG GGT GGT ACT GCT TTT GTT CAA ATG CCA GCT CATGGT TTG TTT CCA TGG TGT GGT TTG TTG TTG GAT ACT AGA ACT TTG GAA GTT CAATCT GAT TAT TCT TCT TAT GCT AGA ACT TCT ATT AGA GCT TCT TTG ACT TTT AATAGA GGT TTT AAA GCT GGT AGA AAT ATG AGA AGA AAA TTG TTT GGT GTT TTG AGATTG AAA TGT CAT TCT TTG TTT TTG GAT TTG CAA GTT AAT TCT TTG CAA ACT GTTTGT ACT AAT ATT TAT AAA ATT TTG TTG TTG CAA GCT TAT AGA TTT CAT GCT TGTGTT TTG CAA TTG CCA TTT CAT CAA CAA GTT TGG AAA AAT CCA ACT TTT TTT TTGAGA GTT ATT TCT GAT ACT GCT TCT TTG TGT TAT TCT ATT TTG AAA GCT AAA AATGCT GGT ATG TCT TTG GGT GCT AAA GGT GCT GCT GGT CCA TTG CCA TCT GAA GCTGTT CAA TGG TTG TGT CAT CAA GCT TTT TTG TTG AAA TTG ACT AGA CAT AGA GTTACT TAT GTT CCA TTG TTG GGT TCT TTG AGA ACT GCT CAA ACT CAA TTG TCT AGAAAA TTG CCA GGT ACT ACT TTG ACT GCT TTG GAA GCT GCT GCT AAT CCA GCT TTGCCA TCT GAT TTT AAA ACT ATT TTG GAT         TABLE 9D Yeast (HighExpressing Genes) (SEQ ID NO:641) ATG CCA AGA GCT CCA AGA TGT AGA GCTGTT AGA TCT TTG TTG AGA TCT CAC TAC AGA GAA GTT TTG CCA TTG GCT ACT TTCGTT AGA AGA TTG GGT CCA CAA GGT TGG AGA TTG GTT CAA AGA GGT GAC CCA GCTGCT TTC AGA GCT TTG GTT GCT CAA TGT TTG GTT TGT GTT CCA TGG GAC GCT AGACCA CCA CCA GCT GCT CCA TCT TTC AGA CAA GTT TCT TGT TTG AAG GAA TTG GTTGCT AGA GTT TTG CAA AGA TTG TGT GAA AGA GGT GCT AAG AAC GTT TTG GCT TTCGGT TTC GCT TTG TTG GAC GGT GCT AGA GGT GGT CCA CCA GAA GCT TTC ACT ACTTCT GTT AGA TCT TAC TTG CCA AAC ACT GTT ACT GAC GCT TTG AGA GGT TCT GGTGCT TGG GGT TTG TTG TTG AGA AGA GTT GGT GAC GAC GTT TTG GTT CAC TTG TTGGCT AGA TGT GCT TTG TTC GTT TTG GTT GCT CCA TCT TGT GCT TAC CAA GTT TGTGGT CCA CCA TTG TAC CAA TTG GGT GCT GCT ACT CAA GCT AGA CCA CCA CCA CACGCT TCT GGT CCA AGA AGA AGA TTG GGT TGT GAA AGA GCT TGG AAC CAC TCT GTTAGA GAA GCT GGT GTT CCA TTG GGT TTG CCA GCT CCA GGT GCT AGA AGA AGA GGTGGT TCT GCT TCT AGA TCT TTG CCA TTG CCA AAG AGA CCA AGA AGA GGT GCT GCTCCA GAA CCA GAA AGA ACT CCA GTT GGT CAA GGT TCT TGG GCT CAC CCA GGT AGAACT AGA GGT CCA TCT GAC AGA GGT TTC TGT GTT GTT TCT CCA GCT AGA CCA GCTGAA GAA GCT ACT TCT TTG GAA GGT GCT TTG TCT GGT ACT AGA CAC TCT CAC CCATCT GTT GGT AGA CAA CAC CAC GCT GGT CCA CCA TCT ACT TCT AGA CCA CCA AGACCA TGG GAC ACT CCA TGT CCA CCA GTT TAC GCT GAA ACT AAG CAC TTC TTG TACTCT TCT GGT GAC AAG GAA CAA TTG AGA CCA TCT TTC TTG TTG TCT TCT TTG AGACCA TCT TTG ACT GGT GCT AGA AGA TTG GTT GAA ACT ATT TTC TTG GGT TCT AGACCA TGG ATG CCA GGT ACT CCA AGA AGA TTG CCA AGA TTG CCA CAA AGA TAC TGGCAA ATG AGA CCA TTG TTC TTG GAA TTG TTG GGT AAC CAC GCT CAA TGT CCA TACGGT GTT TTG TTG AAG ACT CAC TGT CCA TTG AGA GCT GCT GTT ACT CCA GCT GCTGGT GTT TGT GCT AGA GAA AAG CCA CAA GGT TCT GTT GCT GCT CCA GAA GAA GAAGAC ACT GAC CCA AGA AGA TTG GTT CAA TTG TTG AGA CAA CAC TCT TCT CCA TGGCAA GTT TAC GGT TTC GTT AGA GCT TGT TTG AGA AGA TTG GTT CCA CCA GGT TTGTGG GGT TCT AGA CAC AAC GAA AGA AGA TTC TTG AGA AAC ACT AAG AAG TTC ATTTCT TTG GGT AAG CAC GCT AAG TTG TCT TTG CAA GAA TTG ACT TGG AAG ATG TCTGTT AGA GAC TGT GCT TGG TTG AGA AGA TCT CCA GGT GTT GGT TGT GTT CCA GCTGCT GAA CAC AGA TTG AGA GAA GAA ATT TTG GCT AAG TTC TTG CAC TGG TTG ATGTCT GTT TAC GTT GTT GAA TTG TTG AGA TCT TTC TTC TAC GTT ACT GAA ACT ACTTTC CAA AAG AAC AGA TTG TTC TTC TAC AGA AAG TCT GTT TGG TCT AAG TTG CAATCT ATT GGT ATT AGA CAA CAC TTG AAG AGA GTT CAA TTG AGA GAA TTG TCT GAAGCT GAA GTT AGA CAA CAC AGA GAA GCT AGA CCA GCT TTG TTG ACT TCT AGA TTGAGA TTC ATT CCA AAG CCA GAC GGT TTG AGA CCA ATT GTT AAC ATG GAC TAC GTTGTT GGT GCT AGA ACT TTC AGA AGA GAA AAG AGA GCT GAA AGA TTG ACT TCT AGAGTT AAG GCT TTG TTC TCT GTT TTG AAC TAC GAA AGA GCT AGA AGA CCA GGT TTGTTG GGT GCT TCT GTT TTG GGT TTG GAC GAC ATT CAC AGA GCT TGG AGA ACT TTCGTT TTG AGA GTT AGA GCT CAA GAC CCA CCA CCA GAA TTG TAC TTC GTT AAG GTTGAC GTT ACT GGT GCT TAC GAC ACT ATT CCA CAA GAC AGA TTG ACT GAA GTT ATTGCT TCT ATT ATT AAG CCA CAA AAC ACT TAC TGT GTT AGA AGA TAC GCT GTT GTTCAA AAG GCT GCT CAC GGT CAC GTT AGA AAG GCT TTC AAG TCT CAC GTT TCT ACTTTG ACT GAC TTG CAA CCA TAC ATG AGA CAA TTC GTT GCT CAC TTG CAA GAA ACTTCT CCA TTG AGA GAC GCT GTT GTT ATT GAA CAA TCT TCT TCT TTG AAC GAA GCTTCT TCT GGT TTG TTC GAC GTT TTC TTG AGA TTC ATG TGT CAC CAC GCT GTT AGAATT AGA GGT AAG TCT TAC GTT CAA TGT CAA GGT ATT CCA CAA GGT TCT ATT TTGTCT ACT TTG TTG TGT TCT TTG TGT TAC GGT GAC ATG GAA AAC AAG TTG TTC GCTGGT ATT AGA AGA GAC GGT TTG TTG TTG AGA TTG GTT GAC GAC TTC TTG TTG GTTACT CCA CAC TTG ACT CAC GCT AAG ACT TTC TTG AGA ACT TTG GTT AGA GGT GTTCCA GAA TAC GGT TGT GTT GTT AAC TTG AGA AAG ACT GTT GTT AAC TTC CCA GTTGAA GAC GAA GCT TTG GGT GGT ACT GCT TTC GTT CAA ATG CCA GCT CAC GGT TTGTTC CCA TGG TGT GGT TTG TTG TTG GAC ACT AGA ACT TTG GAA GTT CAA TCT GACTAC TCT TCT TAC GCT AGA ACT TCT ATT AGA GCT TCT TTG ACT TTC AAC AGA GGTTTC AAG GCT GGT AGA AAC ATG AGA AGA AAG TTG TTC GGT GTT TTG AGA TTG AAGTGT CAC TCT TTG TTC TTG GAC TTG CAA GTT AAC TCT TTG CAA ACT GTT TGT ACTAAC ATT TAC AAG ATT TTG TTG TTG CAA GCT TAC AGA TTC CAC GCT TGT GTT TTGCAA TTG CCA TTC CAC CAA CAA GTT TGG AAG AAC CCA ACT TTC TTC TTG AGA GTTATT TCT GAC ACT GCT TCT TTG TGT TAC TCT ATT TTG AAG GCT AAG AAC GCT GGTATG TCT TTG GGT GCT AAG GGT GCT GCT GGT CCA TTG CCA TCT GAA GCT GTT CAATGG TTG TGT CAC CAA GCT TTC TTG TTG AAG TTG ACT AGA CAC AGA GTT ACT TACGTT CCA TTG TTG GGT TCT TTG AGA ACT GCT CAA ACT CAA TTG TCT AGA AAG TTGCCA GGT ACT ACT TTG ACT GCT TTG GAA GCT GCT GCT AAC CCA GCT TTG CCA TCTGAC TTC AAG ACT ATT TTG GAC         TABLE 9E “Generic” hTRT ProteinEncoding Sequence (SEQ ID NO:642) ATG CCA CGT GCC CCA CGT TGT CGT GCCGTT CGT TCT TTG TTG CGT TCT CAC TAC CGT GAA GTT TTG CCA TTG GCC ACC TTCGTT CGT CGT TTG GGT CCA CAA GGT TGG CGT TTG GTT CAA CGT GGT GAT CCA GCCGCC TTC CGT GCC TTG GTT GCC CAA TGT TTG GTT TGT GTT CCA TGG GAT GCC CGTCCA CCA CCA GCC GCC CCA TCT TTC CGT CAA GTT TCT TGT TTG AAA GAA TTG GTTGCC CGT GTT TTG CAA CGT TTG TGT GAA CGT GGT GCC AAA AAC GTT TTG GCC TTCGGT TTC GCC TTG TTG GAT GGT GCC CGT GGT GGT CCA CCA GAA GCC TTC ACC ACCTCT GTT CGT TCT TAC TTG CCA AAC ACC GTT ACC GAT GCC TTG CGT GGT TCT GGTGCC TGG GGT TTG TTG TTG CGT CGT GTT GGT GAT GAT GTT TTG GTT CAC TTG TTGGCC CGT TGT GCC TTG TTC GTT TTG GTT GCC CCA TCT TGT GCC TAC CAA GTT TGTGGT CCA CCA TTG TAC CAA TTG GGT GCC GCC ACC CAA GCC CGT CCA CCA CCA CACGCC TCT GGT CCA CGT CGT CGT TTG GGT TGT GAA CGT GCC TGG AAC CAC TCT GTTCGT GAA GCC GGT GTT CCA TTG GGT TTG CCA GCC CCA GGT GCC CGT CGT CGT GGTGGT TCT GCC TCT CGT TCT TTG CCA TTG CCA AAA CGT CCA CGT CGT GGT GCC GCCCCA GAA CCA GAA CGT ACC CCA GTT GGT CAA GGT TCT TGG GCC CAC CCA GGT CGTACC CGT GGT CCA TCT GAT CGT GGT TTC TGT GTT GTT TCT CCA GCC CGT CCA GCCGAA GAA GCC ACC TCT TTG GAA GGT GCC TTG TCT GGT ACC CGT CAC TCT CAC CCATCT GTT GGT CGT CAA CAC CAC GCC GGT CCA CCA TCT ACC TCT CGT CCA CCA CGTCCA TGG GAT ACC CCA TGT CCA CCA GTT TAC GCC GAA ACC AAA CAC TTC TTG TACTCT TCT GGT GAT AAA GAA CAA TTG CGT CCA TCT TTC TTG TTG TCT TCT TTG CGTCCA TCT TTG ACC GQT GCC CGT CGT TTG GTT GAA ACC ATT TTC TTG GGT TCT CGTCCA TGG ATG CCA GGT ACC CCA CGT CGT TTG CCA CGT TTG CCA CAA CGT TAC TGGCAA ATG CGT CCA TTG TTC TTG GAA TTG TTG GGT AAC CAC GCC CAA TGT CCA TACGGT GTT TTG TTG AAA ACC CAC TGT CCA TTG CGT GCC GCC GTT ACC CCA GCC GCCGGT GTT TGT GCC CGT GAA AAA CCA CAA GGT TCT GTT GCC GCC CCA GAA GAA GAAGAT ACC GAT CCA CGT CGT TTG GTT CAA TTG TTG CGT CAA CAC TCT TCT CCA TGGCAA GTT TAC GGT TTC GTT CGT GCC TGT TTG CGT CGT TTG GTT CCA CCA GGT TTGTGG GGT TCT CGT CAC AAC GAA CGT CGT TTC TTG CGT AAC ACC AAA AAA TTC ATTTCT TTG GGT AAA CAC GCC AAA TTG TCT TTG CAA GAA TTG ACC TGG AAA ATG TCTGTT CGT GAT TGT GCC TGG TTG CGT CGT TCT CCA GGT GTT GGT TGT GTT CCA GCCGCC GAA CAC CGT TTG CGT GAA GAA ATT TTG GCC AAA TTC TTG CAC TGG TTG ATGTCT GTT TAC GTT GTT GAA TTG TTG CGT TCT TTC TTC TAC GTT ACC GAA ACC ACCTTC CAA AAA AAC CGT TTG TTC TTC TAC CGT AAA TCT GTT TGG TCT AAA TTG CAATCT ATT GGT ATT CGT CAA CAC TTG AAA CGT GTT CAA TTG CGT GAA TTG TCT GAAGCC GAA GTT CGT CAA CAC CGT GAA GCC CGT CCA GCC TTG TTG ACC TCT CGT TTGCGT TTC ATT CCA AAA CCA GAT GGT TTG CGT CCA ATT GTT AAC ATG GAT TAC GTTGTT GGT GCC CGT ACC TTC CGT CGT GAA AAA CGT GCC GAA CGT TTG ACC TCT CGTGTT AAA GCC TTG TTC TCT GTT TTG AAC TAC GAA CGT GCC CGT CGT CCA GGT TTGTTG GGT GCC TCT GTT TTG GGT TTG GAT GAT ATT CAC CGT GCC TGG CGT ACC TTCGTT TTG CGT GTT CGT GCC CAA GAT CCA CCA CCA GAA TTG TAC TTC GTT AAA GTTGAT GTT ACC GGT GCC TAC GAT ACC ATT CCA CAA GAT CGT TTG ACC GAA GTT ATTGCC TCT ATT ATT AAA CCA CAA AAC ACC TAC TGT GTT CGT CGT TAC GCC GTT GTTCAA AAA GCC GCC CAC GGT CAC GTT CGT AAA GCC TTC AAA TCT CAC GTT TCT ACCTTG ACC GAT TTG CAA CCA TAC ATG CGT CAA TTC GTT GCC CAC TTG CAA GAA ACCTCT CCA TTG CGT GAT GCC GTT GTT ATT GAA CAA TCT TCT TCT TTG AAC GAA GCCTCT TCT GGT TTG TTC GAT GTT TTC TTG CGT TTC ATG TGT CAC CAC GCC GTT CGTATT CGT GGT AAA TCT TAC GTT CAA TGT CAA GGT ATT CCA CAA GGT TCT ATT TTGTCT ACC TTG TTG TGT TCT TTG TGT TAC GGT GAT ATG GAA AAC AAA TTG TTC GCCGGT ATT CGT CGT GAT GGT TTG TTG TTG CGT TTG GTT GAT GAT TTC TTG TTG GTTACC CCA CAC TTG ACC CAC GCC AAA ACC TTC TTG CGT ACC TTG GTT CGT GGT GTTCCA GAA TAC GGT TGT GTT GTT AAC TTG CGT AAA ACC GTT GTT AAC TTC CCA GTTGAA GAT GAA GCC TTG GGT GGT ACC GCC TTC GTT CAA ATG CCA GCC CAC GGT TTGTTC CCA TGG TGT GGT TTG TTG TTG GAT ACC CGT ACC TTG GAA GTT CAA TCT GATTAC TCT TCT TAC GCC CGT ACC TCT ATT CGT GCC TCT TTG ACC TTC AAC CGT GGTTTC AAA GCC GGT CGT AAC ATG CGT CGT AAA TTG TTC GGT GTT TTG CGT TTG AAATGT CAC TCT TTG TTC TTG GAT TTG CAA GTT AAC TCT TTG CAA ACC GTT TGT ACCAAC ATT TAC AAA ATT TTG TTG TTG CAA GCC TAC CGT TTC CAC GCC TGT GTT TTGCAA TTG CCA TTC CAC CAA CAA GTT TGG AAA AAC CCA ACC TTC TTC TTG CGT GTTATT TCT GAT ACC GCC TCT TTG TGT TAC TCT ATT TTG AAA GCC AAA AAC GCC GGTATG TCT TTG GGT GCC AAA GGT GCC GCC GGT CCA TTG CCA TCT GAA GCC GTT CAATGG TTG TGT CAC CAA GCC TTC TTG TTG AAA TTG ACC CGT CAC CGT GTT ACC TACGTT CCA TTG TTG GGT TCT TTG CGT ACC GCC CAA ACC CAA TTG TCT CGT AAA TTGCCA GGT ACC ACC TTG ACC GCC TTG GAA GCC GCC GCC AAC CCA GCC TTG CCA TCTGAT TTC AAA ACC ATT TTG GAT                                

Following determination of the desired nucleotide sequence for the hTRTprotein-encoding polynucleotide, the polynucleotide can be made by anysuitable method including de novo chemical synthesis, directedmutagenesis of a synthetic or naturally occurring TRT gene or cDNA, or acombination of these methods. In one exemplary embodiment,oligonucleotides (typically 50-100 bases in length) are synthesized witha 5′ phosphate group and include approximately 10-base overhangs(relative to adjacent oligonucleotides in the assembled gene) to directsubsequent ligations. Following purification and desalting, eacholigonucleotide is annealed to its complement (e.g., by combining pairsof oligonucleotides in equimolar amounts in a neutral pH buffer with50-200 mM NaCl and 0.5 mM MgCl₂). Annealing may be monitored by nativePAGE. The resulting double-stranded oligonucleotides are ligated totheir neighbors in pairs. After each ligation the products aregel-purified, then ligated to the appropriate (neighboring)double-stranded DNAs. In this manner, fragments of approximately 600-800basepairs are built up. These intermediate fragments are then clonedinto vectors and sequenced. The fragments are then combined into asingle vector (resulting in a vector containing a polynucleotide withthe desired hTRT protein-encoding sequence). This step is facilitated byusing restriction sites present in, or engineered into, thepolynucleotide sequence. Alternatively, the fragments can be built up byligation until the complete cDNA is assembled and the assembled sequencecloned into a vector. Numerous other alternative methods and approacheswill be apparent to those of skill in the art.

Table 10A shows an exemplary set of oligonucleotides that can be used toproduce a polynucleotide, shown in Table 10B, that employs a codondistribution preferentially used by highly expressed genes in E. coli.The sequence in Table 5B contains silent changes to some codons tointroduce useful restriction sites spaced every 300-800 base pairs, tofacilitate subcloning and modification. Oligonucleotide pairs for theinitial annealing steps are indicated by the labels “T” (top strand) and“B” (bottom strand). The full-length polynucleotide (Table 10B) encodesthe hTRT protein (with the start codon at nucleotides 28-30) andcontains Sac I and Xho I sites at the termini flanking the open readingframe, which are useful for cloning into a variety of vectors (e.g.,pBluescript II KS, Stratagene Inc., San Diego Calif.). Once cloned intoan appropriate vector, the hTRT sequence may be expressed, modified(e.g., by site directed or cassette mutagenesis), subcloned, orotherwise used or manipulated. In one embodiment, the polynucleotide issubcloned into a pET vector containing a T7 RNA polymerase promoter(Novagen Inc., Madison, Wis.) and introduced into an E. coli strainhaving an inducible T7 polymerase (Novagen Inc., Madison, Wis.). Oneadvantage to the pET system is that the E. coli culture may be grownbefore the T7 RNA polymerase gene is induced, resulting in very highlevels of transcription and minimizing the effect of any potentialdetrimental effect of the expressed protein on the cells.

TABLE 10 SYNTHESIS OF hTRT POLYNUCLEOTIDE HAVING ALTERNATIVE CODONDISTRIBUTION Table 10A: Oligonucleotides (SEQ ID NOS:643-721) 1BCCAGCGGCAGAACTTCGCGATAGTGGGAACGCAGCAGGGAACGAACAGCACGGCAACGCGGAGCACGCGGCATATGGTCGACTCTAGAGCTCCCGCGTGC 1TGCACGCGGGAGCTCTAGAGTCGACCATATGCCGCGTGCTCCGCGTTGCCGTGCTGTTCGTTCCCTGCTGCGTTCCCACTATCGCGAAGTT 2BGGCACTGAGCAACCAGAGCACGGAAAGCAGCCGGGTCACCACGCTGAACCAGACGCCAACCCTGCGGGCCCAGACGACGAACGAAGGTAG 2TCTGCCGCTGGCTACCTTCGTTCGTCGTCTGGGCCCGCAGGGTTGGCGTCTGGTTCAGCGTGGTGACCCGGCTGCTTTCCGTGCTCTGGTT 3BGAACACGAGCAACCAGTTCTTTCAGGCAGGAAACCTGACGGAAGGACGGAGCAGCCGGCGGCGGACGAGCGTCCCACGGAACGCAAACCA 3TGCTCAGTGCCTGGTTTGCGTTCCGTGGGACGCTCGTCCGCCGCCGGCTGCTCCGTCCTTCCGTCAGGTTTCCTGCCTGAAAGAACTGGTT 4BATGCTTCCGGCGGACCACCACGAGCACCGTCCAGCAGAGCGAAACCGAAAGCCAGAACGTTTTTAGCACCACGTTCGCACAGACGCTGCA 4TGCTCGTGTTCTGCAGCGTCTGTGCGAACGTGGTGCTAAAAACGTTCTGGCTTTCGGTTTCGCTCTGCTGGACGGTGCTCGTGGTGGTCCG 5BCAACACGACGCAGCAGCAGACCCCAAGCACCGGAACCACGCAGAGCGTCGGTAACGGTGTTCGGCAGGTAGGAACGAACGGAGGTGGTGA 5TCCGGAAGCATTCACCACCTCCGTTCGTTCCTACCTGCCGAACACCGTTACCGACGCTCTGCGTGGTTCCGGTGCTTGGGGTCTGCTGCTG 6BGCGGCGGACCACAAACCTGGTAAGCGCAGGACGGAGCAACCAGAACGAACAGAGCGCAACGAGCCAGCAGGTGAACCAGAACGTCGTCAC 6TCGTCGTGTTGGTGACGACGTTCTGGTTCACCTGCTGGCTCGTTGCGCTCTGTTCGTTCTGGTTGCTCCGTCCTGCGCTTACCAGGTTTGT 7BGGTTCCAAGCACGTTCGCAACCCAGACGACGACGCGGACCGGAAGCGTGCGGCGGCGGACGAGCCTGGGTAGCAGCACCCAGCTGGTACA 7TGGTCCGCCGCTGTACCAGCTGGGTGCTGCTACCCAGGCTCGTCCGCCGCCGCACGCTTCCGGTCCGCGTCGTCGTCTGGGTTGCGAACGT 8BGCAGCGGCAGGGAACGGGAAGCGGAACCACCACGACGACGAGCACCCGGAGCCGGCAGACCCAGCGGAACACCAGCTTCACGAACGGAGT 8TGCTTGGAACCACTCCGTTCGTGAAGCTGGTGTTCCGCTGGGTCTGCCGGCTCCGGGTGCTCGTCGTCGTGGTGGTTCCGCTTCCCGTTCC 9BGACCACGGGTACGACCCGGGTGAGCCCAGGAACCCTGACCAACCGGGGTACGTTCCGGTTCCGGAGCAGCACCACGACGCGGACGTTTCG 9TCTGCCGCTGCCGAAACGTCCGCGTCGTGGTGCTGCTCCGGAACCGGAACGTACCCCGGTTGGTCAGGGTTCCTGGGCTCACCCGGGTCGT 10BAGTGACGGGTGCCGGACAGAGCACCTTCCAGGGAGGTAGCTTCTTCAGCCGGACGAGCCGGGGAAACAACGCAGAAACCACGGTCGGACG 10TACCCGTGGTCCGTCCGACCGTGGTTTCTGCGTTGTTTCCCCGGCTCGTCCGGCTGAAGAAGCTACCTCCCTGGAAGGTGCTCTGTCCGGC 11BAAACCGGCGGGCACGGGGTGTCCCACGGACGCGGCGGACGGGAGGTGGACGGCGGACCAGCGTGGTGCTGACGACCAACGGACGGGTGGG 11TACCCGTCACTCCCACCCGTCCGTTGGTCGTCAGCACCACGCTGGTCCGCCGTCCACCTCCCGTCCGCCGCGTCCGTGGGACACCCCGTGC 12BTCAGGGACGGACGCAGGGAGGACAGCAGGAAGGACGGACGCAGCTGTTCTTTGTCACCGGAGGAGTACAGGAAGTGTTTGGTTTCAGCGT 12TCCGCCGGTTTACGCTGAAACCAAACACTTCCTGTACTCCTCCGGTGACAAAGAACAGCTGCGTCCGTCCTTCCTGCTGTCCTCCCTGCGT 13BGCTGCGGCAGACGCGGCAGACGACGCGGGGTGCCCGGCATCCACGGACGGGAACCCAGGAAGATAGTTTCAACCAGACGACGAGCACCGG 13TCCGTCCCTGACCGGTGCTCGTCGTCTGGTTGAAACTATCTTCCTGGGTTCCCGTCCGTGGATGCCGGGCACCCCGCGTCGTCTGCCGCGT 14BGCGGGCAGTGGGTTTTCAGCAGAACACCATACGGGCACTGAGCGTGGTTGCCCAGCAGTTCCAGGAACAGCGGACGCATCTGCCAGTAAC 14TCTGCCGCAGCGTTACTGGCAGATGCGTCCGCTGTTCCTGGAACTGCTGGGCAACCACGCTCAGTGCCCGTATGGTGTTCTGCTGAAAACC 15BGGTCGGTATCTTCTTCTTCCGGAGCAGCAACGGAACCCTGCGGTTTTTCACGAGCGCAAACACCAGCAGCCGGGGTAACAGCAGCACGCA 15TCACTGCCCGCTGCGTGCTGCTGTTACCCCGGCTGCTGGTGTTTGCGCTCGTGAAAAACCGCAGGGTTCCGTTGCTGCTCCGGAAGAAGAA 16BGCGGAACCAGACGACGCAGGCATGCACGAACGAAACCGTAAACCTGCCACGGGGAGGAGTGCTGACGCAGCAGCTGAACCAGACGACGCG 16TGATACCGACCCGCGTCGTCTGGTTCAGCTGCTGCGTCAGCACTCCTCCCCGTGGCAGGTTTACGGTTTCGTTCGTGCATGCCTGCGTCGT 17BGGGACAGTTTAGCGTGTTTACCCAGGGAGATGAATTTTTTGGTGTTACGCAGGAAACGACGTTCGTTGTGACGGGAACCCCACAGACCCG 17TCTGGTTCCGCCGGGTCTGTGGGGTTCCCGTCACAACGAACGTCGTTTCCTGCGTAACACCAAAAAATTCATCTCCCTGGGTAAACACGCT 18BGGTGTTCAGCAGCCGGAACGCAACCAACACCCGGAGAACGACGCAGCCAAGCGCAGTCACAACGGACATTTTCCAGGTCAGTTCCTGCA 18TAAACTGTCCCTGCAGGAACTGACCTGGAAAATGTCCGTTCGTGACTGCGCTTGGCTGCGTCGTTCTCCGGGTGTTGGTTGCGTTCCGGCT 19BCGGTAACGTAGAAGAAGGAACGCAGCAGTTCAACAACGTATACGGACATCAGCCAGTGCAGGAATTTAGCCAGGATTTCTTCACGCAGAC 19TGCTGAACACCGTCTGCGTGAAGAAATCCTGGCTAAATTCCTGCACTGGCTGATGTCCGTATACGTTGTTGAACTGCTGCGTTCCTTCTTC 20BGTTTCAGGTGCTGACGGATACCGATGGACTGCAGTTTGGACCAAACGGATTTACGGTAGAAGAACAGACGGTTTTTCTGGAAGGTGGTTT 20TTACGTTACCGAAACCACCTTCCAGAAAAACCGTCTGTTCTTCTACCGTAAATCCGTTTGGTCCAAACTGCAGTCCATCGGTATCCGTCAG 21BGATGAAACGCAGACGGGAGGTCAGCAGAGCCGGACGAGCTTCACGGTGCTGACGAACTTCAGCTTCGGACAGTTCACGCAGCTGAACAC 21TCACCTGAAACGTGTTCAGCTGCGTGAACTGTCCGAAGCTGAAGTTCGTCAGCACCGTGAACTCGTCCGGCTCTGCTGACCTCCCGTCTG 22BTCAGACGCTCAGCACGTTTTTCACGACGGAAGGTACGAGCACCAACAACGTAGTCCATGTTTACGATCGGACGCAGACCGTCCGGTTTCG 22TCGTTTCATCCCGAAACCGGACGGTCTGCGTCCGATCGTAAACATGGACTACGTTGTTGGTGCTCGTACCTTCCGTCGTGAAAAACGTGCT 23BCGTCCAGACCCAGAACGGAAGCACCCAGCAGACCCGGACGACGAGCACGTTCGTAGTTCAGAACGGAGAACAGAGCTTTAACACGGGAGG 23TGAGCGTCTGACCTCCCGTGTTAAAGCTCTGTTCTCCGTTCTGAACTACGAACGTGCTCGTCGTCCGGGTCTGCTGGGTGCTTCCGTTCTG 24BCGGTAACGTCAACTTTAACGAAGTACAGTTCCGGCGGCGGGTCCTGAGCACGAACACGCAGAACGAAGGTACGCCAAGCACGGTGGATGT 24TGGTCTGGACGACATCCACCGTGCTTGGCGTACCTTCGTTCTGCGTGTTCGTGCTCAGGACCCGCCGCCGGAACTGTACTTCGTTAAAGTT 25BCGTAACGACGAACGCAGTAGGTGTTCTGCGGTTTGATGATGGAAGCGATAACTTCGGTCAGACGGTCCTGCGGGATGGTGTCGTACGCGC 25TGACGTTACCGGCGCGTACGACACCATCCCGCAGGACCGTCTGACCGAAGTTATCGCTTCCATCATCAAACCGCAGAACACCTACTGCGTT 26BGACGCATGTACGGCTGCAGGTCGGTCAGGGTGGAAACGTGGGATTTGAATGCTTTACGAACGTGACCGTGAGCAGCTTTCTGAACAACAG 26TCGTCGTTACGCTGTTGTTCAGAAAGCTGCTCACGGTCACGTTCGTAAAGCATTCAAATCCCACGTTTCCACCCTGACCGACCTGCAGCCG 27BGACCGGAGGAAGCTTCGTTCAGGGAGGAGGACTGTTCGATAACAACAGCGTCACGCAGCGGGGAGGTTTCCTGCAGGTGAGCAACGAACT 27TTACATGCGTCAGTTCGTTGCTCACCTGCAGGAAACCTCCCCGCTGCGTGACGCTGTTGTTATCGAACAGTCCTCCTCCCTGAACGAAGCT 28BAACCCTGCGGGATACCCTGGCACTGAACGTAGGATTTACCACGGATACGAACAGCGTGGTGGCACATGAAACGCAGGAAAACGTCGAACA 28TTCCTCCGGTCTGTTCGACGTTTTCCTGCGTTTCATGTGCCACCACGCTGTTCGTATCCGTGGTAAATCCTACGTTCAGTGCCAGGGTATC 29BGCAGCAGCAGACCGTCACGACGGATACCAGCGAACAGTTTGTTTTCCATGTCACCGTAGCACAGGGAGCACAGCAGGGTGGACAGGATGG 29TCCGCAGGGTTCCATCCTGTCCACCCTGCTGTGCTCCCTGTGCTACGGTGACATGGAAAACAAACTGTTCGCTGGTATCCGTCGTGACGGT 30BCGTATTCCGGAACACCACGAACCAGGGTACGCAGGAAGGTTTTAGCGTGGGTCAGGTGCGGAGTAACCAGCAGGAAGTCGTCAACCAGAC 30TCTGCTGCTGCGTCTGGTTGACGACTTCCTGCTGGTTACTCCGCACCTGACCCACGCTAAAACCTTCCTGCGTACCCTGGTTCGTGGTGTT 31BGAGCCGGCATCTGAACGAAAGCGGTGCCACCCAGAGCTTCGTCTTCAACCGGGAAGTTAACAACGGTTTTACGCAGGTTTACAACGCAAC 31TCCGGAATACGGTTGCGTTGTAAACCTGCGTAAAACCGTTGTTAACTTCCCGGTTGAAGACGAAGCTCTGGGTGGCACCGCTTTCGTTCAG 32BGGATGGAGGTACGAGCGTAGGAGGAGTAGTCGGACTGAACTTCCAGGGTACGGGTGTCCAGCAGCAGACCGCACCACGGGAACAGACCGT 32TATGCCGGCTCACGGTCTGTTCCCGTGGTGCGGTCTGCTGCTGGACACCCGTACCCTGGAAGTTCAGTCCGACTACTCCTCCTACGCTCGT 33BGGGAGTGGCATTTCAGACGCAGAACACCGAACAGTTTACGACGCATGTTACGACCAGCTTTGAAACCACGGTTGAAGGTCAGGGAAGCAC 33TACCTCCATCCGTGCTTCCCTGACCTTCAACCGTGGTTTCAAAGCTGGTCGTAACATGCGTCGTAAACTGTTCGGTGTTCTGCGTCTGAAA 34BACGCGTGGAAACGGTAAGCCTGCAGCAGCAGGATTTTGTAGATGTTGGTGCAAACGGTCTGCAGGGAGTTTACCTGCAGGTCCAGGAACA 34TTGCCACTCCCTGTTCCTGGACCTGCAGGTAAACTCCCTGCAGACCGTTTGCACCAACATCTACAAAATCCTGCTGCTGCAGGCTTACCGT 35BAGTAGCACAGGGAAGCGGTGTCGGAGATAACACGCAGGAAGAAGGTCGGGTTTTTCCAAACCTGCTGGTGGAACGGCAGCTGCAGAACGC 35TTTCCACGCGTGCGTTCTGCAGCTGCCGTTCCACCAGCAGGTTTGGAAAAACCCGACCTTCTTCCTGCGTGTTATCTCCGACACCGCTTCC 36BGGCACAGCCACTGAACAGCTTCGGACGGCAGCGGACCAGCAGCACCTTTAGCACCCAGGGACATACCAGCGTTTTTAGCTTTCAGGATGG 36TCTGTGCTACTCCATCCTGAAAGCTAAAAACGCTGGTATGTCCCTGGGTGCTAAAGGTGCTGCTGGTCCGCTGCCGTCCGAAGCTGTTCAG 37BACAGCTGGGTCTGAGCGGTACGCAGGGAACCCAGCAGCGGAACGTAGGTAACACGGTGACGGGTCAGTTTCAGCAGGAAAGCCTGGT 37TTGGCTGTGCCACCAGGCTTTCCTGCTGAAACTGACCCGTCACCGTGTTACCTACGTTCCGCTGCTGGGTTCCCTGCGTACCGCTCAG 38BACGGCAGAGCCGGGTTAGCAGCAGCTTCCAGAGCGGTCAGGGTGGTACCCGGCAGTTTACG GG 38TACCCAGCTGTCCCGTAAACTGCCGGGTACCACCCTGACCGCTCTGGAAGCTGCTGCTAACCC GG 39BGCGTGCCTCGAGGAATTCGGATCCATTAGTCCAGGATGGTTTTGAAGTCG 39TCTCTGCCGTCCGACTTCAAAACCATCCTGGACTAATGGATCCGAATTCCTCGAGGCACGC Table 10B(SEQ ID NO:721)GCACGCGGGAGCTCTAGAGTCGACCATATGCCGCGTGCTCCGCGTTGCCGTGCTGTTCGTTCCCTGCTGCGTTCCCACTATCGCGAAGTTCTGCCGCTGGCTACCTTCGTTCGTCGTCTGGGCCCGCAGGGTTGGCGTCTGGTTCAGCGTGGTGACCCGGCTGCTTTCCGTGCTCTGGTTGCTCAGTGCCTGGTTTGCGTTCCGTGGGACGCTCGTCCGCCGCCGGCTGCTCCGTCCTTCCGTCAGGTTTCCTGCCTGAAAGAACTGGTTGCTCGTGTTCTGCAGCGTCTGTGCGAACGTGGTGCTAAAAACGTTCTGGCTTTCGGTTTCGCTCTGCTGGACGGTGCTCGTGGTGGTCCGCCGGAAGCATTCACCACCTCCGTTCGTTCCTACCTGCCGAACACCGTTACCGACGCTCTGCGTGGTTCCGGTGCTTGGGGTCTGCTGCTGCGTCGTGTTGGTGACGACGTTCTGGTTCACCTGCTGGCTCGTTGCGCTCTGTTCGTTCTGGTTGCTCCGTCCTGCGCTTACCAGGTTTGTGGTCCGCCGCTGTACCAGCTGGGTGCTGCTACCCAGGCTCGTCCGCCGCCGCACGCTTCCGGTCCGCGTCGTCGTCTGGGTTGCGAACGTGCTTGGAACCACTCCGTTCGTGAAGCTGGTGTTCCGCTGGGTCTGCCGGCTCCGGGTGCTCGTCGTCGTGGTGGTTCCGCTTCCCGTTCCCTGCCGCTGCCGAAACGTCCGCGTCGTGGTGCTGCTCCGGAACCGGAACGTACCCCGGTTGGTCAGGGTTCCTGGGCTCACCCGGGTCGTACCCGTGGTCCGTCCGACCGTGGTTTCTGCGTTGTTTCCCCGGCTCGTCCGGCTGAAGAAGCTACCTCCCTGGAAGGTGCTCTGTCCGGCACCCGTCACTCCCACCCGTCCGTTGGTCGTCAGCACCACGCTGGTCCGCCGTCCACCTCCCGTCCGCCGCGTCCGTGGGACACCCCGTGCCCGCCGGTTTACGCTGAAACCAAACACTTCCTGTACTCCTCCGGTGACAAAGAACAGCTGCGTCCGTCCTTCCTGCTGTCCTCCCTGCGTCCGTCCCTGACCGGTGCTCGTCGTCTGGTTGAAACTATCTTCCTGGGTTCCCGTCCGTGGATGCCGGGCACCCCGCGTCGTCTGCCGCGTCTGCCGCAGCGTTACTGGCAGATGCGTCCGCTGTTCCTGGAACTGCTGGGCAACCACGCTCAGTGCCCGTATGGTGTTCTGCTGAAAACCCACTGCCCGCTGCGTGCTGCTGTTACCCCGGCTGCTGGTGTTTGCGCTCGTGAAAAACCGCAGGGTTCCGTTGCTGCTCCGGAAGAAGAAGATACCGACCCGCGTCGTCTGGTTCAGCTGCTGCGTCAGCACTCCTCCCCGTGGCAGGTTTACGGTTTCGTTCGTGCATGCCTGCGTCGTCTGGTTCCGCCGGGTCTGTGGGGTTCCCGTCACAACGAACGTCGTTTCCTGCGTAACACCAAAAAATTCATCTCCCTGGGTAAACACGCTAAACTGTCCCTGCAGGAACTGACCTGGAAAATGTCCGTTCGTGACTGCGCTTGGCTGCGTCGTTCTCCGGGTGTTGGTTGCGTTCCGGCTGCTGAACACCGTCTGCGTGAAGAAATCCTGGCTAAATTCCTGCACTGGCTGATGTCCGTATACGTTGTTGAACTGCTGCGTTCCTTCTTCTACGTTACCGAAACCACCTTCCAGAAAAACCGTCTGTTCTTCTACCGTAAATCCGTTTGGTCCAAACTGCAGTCCATCGGTATCCGTCAGCACCTGAAACGTGTTCAGCTGCGTGAACTGTCCGAAGCTGAAGTTCGTCAGCACCGTGAAGCTCGTCCGGCTCTGCTGACCTCCCGTCTGCGTTTCATCCCGAAACCGGACGGTCTGCGTCCGATCGTAAACATGGACTACGTTGTTGGTGCTCGTACCTTCCGTCGTGAAAAACGTGCTGAGCGTCTGACCTCCCGTGTTAAAGCTCTGTTCTCCGTTCTGAACTACGAACGTGCTCGTCGTCCGGGTCTGCTGGGTGCTTCCGTTCTGGGTCTGGACGACATCCACCGTGCTTGGCGTACCTTCGTTCTGCGTGTTCGTGCTCAGGACCCGCCGCCGGAACTGTACTTCGTTAAAGTTGACGTTACCGGCGCGTACGACACCATCCCGCAGGACCGTCTGACCGAAGTTATCGCTTCCATCATCAAACCGCAGAACACCTACTGCGTTCGTCGTTACGCTGTTGTTCAGAAAGCTGCTCACGGTCACGTTCGTAAAGCATTCAAATCCCACGTTTCCACCCTGACCGACCTGCAGCCGTACATGCGTCAGTTCGTTGCTCACCTGCAGGAAACCTCCCCGCTGCGTGACGCTGTTGTTATCGAACAGTCCTCCTCCCTGAACGAAGCTTCCTCCGGTCTGTTCGACGTTTTCCTGCGTTTCATGTGCCACCACGCTGTTCGTATCCGTGGTAAATCCTACGTTCAGTGCCAGGGTATCCCGCAGGGTTCCATCCTGTCCACCCTGCTGTGCTCCCTGTGCTACGGTGACATGGAAAACAAACTGTTCGCTGGTATCCGTCGTGACGGTCTGCTGCTGCGTCTGGTTGACGACTTCCTGCTGGTTACTCCGCACCTGACCCACGCTAAAACCTTCCTGCGTACCCTGGTTCGTGGTGTTCCGGAATACGGTTGCGTTGTAAACCTGCGTAAAACCGTTGTTAACTTCCCGGTTGAAGACGAAGCTCTGGGTGGCACCGCTTTCGTTCAGATGCCGGCTCACGGTCTGTTCCCGTGGTGCGGTCTGCTGCTGGACACCCGTACCCTGGAAGTTCAGTCCGACTACTCCTCCTACGCTCGTACCTCCATCCGTGCTTCCCTGACCTTCAACCGTGGTTTCAAAGCTGGTCGTAACATGCGTCGTAAACTGTTCGGTGTTCTGCGTCTGAAATGCCACTCCCTGTTCCTGGACCTGCAGGTAAACTCCCTGCAGACCGTTTGCACCAACATCTACAAAATCCTGCTGCTGCAGGCTTACCGTTTCCACGCGTGCGTTCTGCAGCTGCCGTTCCACCAGCAGGTTTGGAAAAACCCGACCTTCTTCCTGCGTGTTATCTCCGACACCGCTTCCCTGTGCTACTCCATCCTGAAAGCTAAAAACGCTGGTATGTCCCTGGGTGCTAAAGGTGCTGCTGGTCCGCTGCCGTCCGAAGCTGTTCAGTGGCTGTGCCACCAGGCTTTCCTGCTGAAACTGACCCGTCACCGTGTTACCTACGTTCCGCTGCTGGGTTCCCTGCGTACCGCTCAGACCCAGCTGTCCCGTAAACTGCCGGGTACCACCCTGACCGCTCTGGAAGCTGCTGCTAACCCGGCTCTGCCGTCCGACTTCAAAACCATCCTGGACTAATGGATCCGAATTCCTCGAGGCACGC

The present invention also provides transgenic animals (i.e., mammalstransgenic for a human or other TRT gene sequence) expressing an hTRT orother TRT polynucleotide or polypeptide. In one embodiment, hTRT issecreted into the milk of a transgenic mammal such as a transgenicbovine, goat, or rabbit. Methods for production of such animals arefound, e.g., in Heyneker et al., PCT WO 91/08216.

The hTRT proteins and complexes of the invention, including those madeusing the expression systems disclosed herein supra, may be purifiedusing a variety of general methods known in the art in accordance withthe specific methods provided by the present invention (e.g., infra).One of skill in the art will recognize that after chemical synthesis,biological expression, or purification, the hTRT protein may possess aconformation different than a native conformation of naturally occurringtelomerase. In some instances, it may be helpful or even necessary todenature (e.g., including reduction of disulfide or other linkages) thepolypeptide and then to cause the polypeptide to re-fold into thepreferred conformation. Productive refolding may also require thepresence of hTR (or hTR fragments). Methods of reducing and denaturingproteins and inducing re-folding are well known to those of skill in theart (see, e.g., Debinski et al., 1993, J. Biol. Chem., 268:14065;Kreitman and Pastan, 1993, Bioconjug. Chem., 4:581; and Buchner et al.,1992, Anal. Biochem., 205:263; and McCaman et al., 1985, J. Biotech.2:177). See also PCT Publication WO 96/40868, supra.

D) Complexes of Human TRT and Human Telomerase RNA,Telomerase-Associated Proteins, and Other Biomolecules Produced byCoexpression and Other Means

hTRT polypeptides of the invention can associate in vivo and in vitrowith other biomolecules, including RNAs (e.g., hTR), proteins (e.g.,telomerase-associated proteins), DNA (e.g., telomeric DNA, [T₂AG₃]_(N)),and nucleotides, such as (deoxy)ribonucleotide triphosphates. Theseassociations can be exploited to assay hTRT presence or function, toidentify or purify hTRT or telomerase-associated molecules, and toanalyze hTRT or telomerase structure or function in accordance with themethods of the present invention.

In one embodiment, the present invention provides hTRT complexed with(e.g., associated with or bound to) a nucleic acid, usually an RNA, forexample to produce a telomerase holoenzyme. In one embodiment, the boundRNA is capable of acting as a template for telomerase-mediated DNAsynthesis. Examples of RNAs that may be complexed with the hTRTpolypeptide include a naturally occurring host cell telomerase RNA, ahuman telomerase RNA (e.g., hTR; U.S. Pat. No. 5,583,016), an hTRsubsequence or domain, a synthetic RNA, or other RNAs. The RNA-hTRTprotein complex (an RNP) typically exhibits one or more telomeraseactivities, such as telomerase catalytic activities. These hTRT-hTR RNPs(or other hTRT-RNA complexes) can be produced by a variety of methods,as described infra for illustrative purposes, including in vitroreconstitution, by co-expression of hTRT and hTR (or other RNA) in vitro(i.e., in a cell free system), in vivo reconstitution, or ex vivoreconstitution.

Thus, the present invention provides, in one embodiment, an hTRT-hTRcomplex (or other hTRT-RNA complex) formed in vitro by mixing separatelypurified components (“in vitro reconstitution;” see, e.g., U.S. Pat. No.5,583,016 for a description of reconstitution; also see Autexier et al.,EMBO J. 15:5928). In one embodiment the hTRT protein is produced byrecombinant expression in human or non-human cells, e.g., as describedsupra, and subsequently purified using protein purification methods(e.g., chromatography, affinity purification). In a particularembodiment, the recombinant hTRT protein is purified to homogeneity. Thepurified hTRT protein is combined with separately purified hTR, whichmay be produced using an in vitro transcription system, by chemicalsynthesis, or by other methods and purified using standard RNApurification techniques (see Melton et al., 1984, Nucl. Acids Res.12:7035; Studier et al., 1986, J. Mol. Biol. 189:113).

In an alternative embodiment, the invention provides telomerase RNPsproduced by coexpression of the hTRT polypeptide and an RNA (e.g., hTR)in vitro in a cell-free transcription-translation system (e.g. wheatgerm or rabbit reticulocyte lysate). As shown in Example 7, in vitroco-expression of a recombinant hTRT polypeptide and hTR results inproduction of telomerase catalytic activity (as measured by a TRAPassay).

Further provided by the present invention are telomerase RNPs producedby expression of the hTRT polypeptide in a cell, e.g., a mammalian cell,in which hTR is naturally expressed or in which hTR (or another RNAcapable of forming a complex with the hTRT protein) is introduced orexpressed by recombinant means. Thus, in one embodiment, hTRT isexpressed in a telomerase negative human cell in which hTR is present(e.g., BJ or IMP90 cells), allowing the two molecules to assemble intoan RNP. In another embodiment, hTRT is expressed in a human or non-humancell in which hTR is recombinantly expressed. Methods for expression ofhTR in a cell are found in U.S. Pat. No. 5,583,016. Further, a clonecontaining a cDNA encoding the RNA component of telomerase has beenplaced on deposit as pGRN33 (ATCC 75926). Genomic sequences encoding theRNA component of human telomerase are also on deposit in the ˜15 kbSauIIIA1 to HindIII insert of lambda clone 28-1 (ATCC 75925). Forexpression in eukaryotic cells the hTRT sequence will typically beoperably linked to a transcription initiation sequence (RNA polymerasebinding site) and transcription terminator sequences (see, e.g., PCTPublication WO 96/01835; Feng et al., 1995, Science 269:1236).

The present invention further provides recombinantly produced orsubstantially purified hTRT polypeptides coexpressed and/or associatedwith so-called “telomerase-associated proteins.” Thus, the presentinvention provides hTRT coexpressed with, or complexed with, otherproteins (e.g., telomerase-associated proteins). Telomerase-associatedproteins are those proteins that copurify with human telomerase and/orthat may play a role in modulating telomerase function or activity, forexample by participating in the association of telomerase with telomericDNA. Examples of telomerase-associated proteins include (but are notlimited to) the following proteins and/or their human homologs:nucleolin (see, Srivastava et al., 1989, FEBS Letts. 250:99); EF2H(elongation factor 2 homolog; see Nomura et al. 1994, DNA Res. (Japan)1:27, GENBANK accession #D21163); TP1/TLP1 (Harrington et al., 1997,Science 275:973; Nakayama, 1997, Cell 88:875); the human homologue ofthe Tetrahymena p95 or p95 itself (Collins et al., 1995, Cell 81:677);TPC2 (a telomere length regulatory protein; ATCC accession number 97708;TPC3 (also a telomere length regulatory protein; ATCC accession number97707; DNA-binding protein B (dbpB; Horwitz et al., 1994, J. Biol. Chem.269:14130; and Telomere Repeat Binding Factors (TRF 1 & 2; Chang et al.,1995, Science 270:1663; Chong et al., 1997, Hum Mol Genet 6:69); EST1, 3and 4 (Lendvay et al., 1996, Genetics 144:1399, Nugent et al., 1996,Science 274:249, Lundblad et al., 1989, Cell 57:633); and End-cappingfactor (Cardenas et al., 1993, Genes Dev. 7:883).

Telomerase associated proteins can be identified on the basis ofco-purification with, or binding to, hTRT protein or the hTRT-hTR RNP.Alternatively, they can be identified on the basis of binding to an hTRTfusion protein, e.g., a GST-hTRT fusion protein or the like, asdetermined by affinity purification (see, Ausubel et al. Ch 20). Aparticularly useful technique for assessing protein-proteininteractions, which is applicable to identifying hTRT-associatedproteins, is the two hybrid screen method of Chien et al. (Proc. Natl.Acad. Sci. USA 88:9578 [1991]; see also Ausubel et al., supra, at Ch.20). This screen identifies protein-protein interactions in vivo throughreconstitution of a transcriptional activator, the yeast Gal4transcription protein (see, Fields and Song, 1989, Nature 340:245). Themethod is based on the properties of the yeast Gal4 protein, whichconsists of separable domains responsible for DNA-binding andtranscriptional activation. Polynucleotides, usually expression vectors,encoding two hybrid proteins are constructed. One polynucleotidecomprises the yeast Gal4 DNA-binding domain fused to a polypeptidesequence of a protein to be tested for an hTRT interaction (e.g.,nucleolin or EF2H). Alternatively the yeast Gal4 DNA-binding domain isfused to cDNAs from a human cell, thus creating a library of humanproteins fused to the Gal4 DNA binding domain for screening fortelomerase associated proteins. The other polynucleotide comprises theGal4 activation domain fused to an hTRT polypeptide sequence. Theconstructs are introduced into a yeast host cell. Upon expression,intermolecular binding between hTRT and the test protein canreconstitute the Gal4 DNA-binding domain with the Gal4 activationdomain. This leads to the transcriptional activation of a reporter gene(e.g., lacZ, HIS3) operably linked to a Gal4 binding site. By selectingfor, or by assaying the reporter, gene colonies of cells that contain anhTRT interacting protein or telomerase associated protein can beidentified. Those of skill will appreciate that there are numerousvariations of the 2-hybrid screen, e.g., the LexA system (Bartel et al,1993, in Cellular Interactions in Development: A Practical Approach Ed.Hartley, D. A. (Oxford Univ. Press) pp. 153-79).

Another useful method for identifying telomerase-associated proteins isa three-hybrid system (see, e.g., Zhang et al., 1996, Anal. Biochem.242:68; Licitra et al., 1996, Proc. Natl. Acad. Sci. USA 93:12817). Thetelomerase RNA component can be utilized in this system with the TRT orhTRT protein and a test protein. Another useful method for identifyinginteracting proteins, particularly (i.e., proteins that heterodimerizeor form higher order heteromultimers), is the E. coli/BCCP interactivescreening system (see, Germino et al. (1993) Proc. Natl. Acad. Sci.U.S.A. 90:933; Guarente (1993) Proc. Natl. Acad. Sci. (U.S.A.) 90:1639).

The present invention also provides complexes of telomere bindingproteins (which may or may not be telomerase associated proteins) andhTRT (which may or may not be complexed with hTR, other RNAs, or one ormore telomerase associated proteins). Examples of telomere bindingproteins include TRF1 and TRF2 (supra); rnpA1, rnpA2, RAP1 (Buchman etal., 1988, Mol. Cell. Biol. 8:210, Buchman et al., 1988, Mol. Cell.Biol. 8:5086), SIR3 and SIR4 (Aparicio et al, 1991, Cell 66:1279), TEL1(Greenwell et al., 1995, Cell 82:823; Morrow et al., 1995, Cell 82:831);ATM (Savitsky et al., 1995, Science 268:1749), end-capping factor(Cardenas et al., 1993, Genes Dev. 7:883), and corresponding humanhomologs. The aforementioned complexes may be produced generally asdescribed supra for complexes of hTRT and hTR or telomerase associatedproteins, e.g., by mixing or co-expression in vitro or in vivo.

V. Antibodies and Other Binding Agents

In a related aspect, the present invention provides antibodies that arespecifically immunoreactive with hTRT, including polyclonal andmonoclonal antibodies, antibody fragments, single chain antibodies,human and chimeric antibodies, including antibodies or antibodyfragments fused to phage coat or cell surface proteins, and others knownin the art and described herein. The antibodies of the invention canspecifically recognize and bind polypeptides that have an amino acidsequence that is substantially identical to the amino acid sequence setforth in FIG. 17 SEQ ID NO:2, or an immunogenic fragment thereof orepitope on the protein defined thereby. The antibodies of the inventioncan exhibit a specific binding affinity for hTRT of at least about 10⁷,10⁸, 10⁹, or 10¹⁰ M⁻¹, and may be polyclonal, monoclonal, recombinant orotherwise produced. The invention also provides anti-hTRT antibodiesthat recognize an hTRT conformational epitope (e.g., an epitope on thesurface of the hTRT protein or a telomerase RNP). Likely conformationalepitopes can be identified, if desired, by computer-assisted analysis ofthe hTRT protein sequence, comparison to the conformation of relatedreverse transcriptases, such as the p66 subunit of HIV-1 (see, e.g.,FIG. 3), or empirically. Anti-hTRT antibodies that recognizeconformational epitopes have utility, inter alia, in detection andpurification of human telomerase and in the diagnosis and treatment ofhuman disease.

For the production of anti-hTRT antibodies, hosts such as goats, sheep,cows, guinea pigs, rabbits, rats, or mice, may be immunized by injectionwith hTRT protein or any portion, fragment or oligopeptide thereof whichretains immunogenic properties. In selecting hTRT polypeptides forantibody induction, one need not retain biological activity; however,the protein fragment, or oligopeptide must be immunogenic, andpreferably antigenic. Immunogenicity can be determined by injecting apolypeptide and adjuvant into an animal (e.g., a rabbit) and assayingfor the appearance of antibodies directed against the injectedpolypeptide (see, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York (1988), which isincorporated in its entirety and for all purposes, e.g., at Chapter 5).Peptides used to induce specific antibodies typically have an amino acidsequence consisting of at least five amino acids, preferably at least 8amino acids, more preferably at least 10 amino acids. Usually they willmimic or have substantial sequence identity to all or a contiguousportion of the amino acid sequence of the protein of SEQ ID NO:2. Shortstretches of hTRT protein amino acids may be fused with those of anotherprotein, such as keyhole limpet hemocyanin, and an anti-hTRT antibodyproduced against the chimeric molecule. Depending on the host species,various adjuvants may be used to increase immunological response.

The antigen is presented to the immune system in a fashion determined bymethods appropriate for the animal. These and other parameters aregenerally well known to immunologists. Typically, injections are givenin the footpads, intramuscularly, intradermally, perilymph nodally orintraperitoneally. The immunoglobulins produced by the host can beprecipitated, isolated and purified by routine methods, includingaffinity purification.

Illustrative examples of immunogenic hTRT peptides include are providedin Example 8. In addition, Example 8 describes the production and use ofanti-hTRT polyclonal antibodies.

A) Monoclonal Antibodies

Monoclonal antibodies to hTRT proteins and peptides may be prepared inaccordance with the methods of the invention using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique originally described by Koehler and Milstein (Nature 256:495[1975]), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunol. Today 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci. USA,80:2026), and the EBV-hybridoma technique (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R Liss Inc, New York N.Y., pp 77-96[1985]).

In one embodiment, appropriate animals are selected and the appropriateimmunization protocol followed. The production of non-human monoclonalantibodies, e.g., murine, lagomorpha, equine, is well known and can beaccomplished by, for example, immunizing an animal with a preparationcontaining hTRT or fragments thereof. In one method, after theappropriate period of time, the spleens of the animals are excised andindividual spleen cells are fused, typically, to immortalized myelomacells under appropriate selection conditions. Thereafter, the cells areclonally separated and the supernatants of each clone (e.g., hybridoma)are tested for the production of an appropriate antibody specific forthe desired region of the antigen. Techniques for producing antibodiesare well known in the art. See, e.g., Goding et al., MonoclonalAntibodies: Principles and Practice (2d ed.) Acad. Press, N.Y., andHarlow and Lane, supra, each of which is incorporated in its entiretyand for all purposes. Other suitable techniques involve the in vitroexposure of lymphocytes to the antigenic polypeptides or alternatively,to selection of libraries of antibodies in phage or similar vectors(see, infra).

B) Human Antibodies

In another aspect of the invention, human antibodies against an hTRTpolypeptide are provided. Human monoclonal antibodies against a knownantigen can also be made using transgenic animals having elements of ahuman immune system (see, e.g., U.S. Pat. Nos. 5,569,825 and 5,545,806,both of which are incorporated by reference in their entirety for allpurposes) or using human peripheral blood cells (Casali et al., 1986,Science 234:476). Some human antibodies are selected by competitivebinding experiments, or otherwise, to have the same epitope specificityas a particular mouse antibody.

In an alternative embodiment, human antibodies to an hTRT polypeptidecan be produced by screening a DNA library from human B cells accordingto the general protocol outlined by Huse et al., 1989, Science 246:1275,which is incorporated by reference. Antibodies binding to the hTRTpolypeptide are selected. Sequences encoding such antibodies (or bindingfragments) are then cloned and amplified. The protocol described by Huseis often used with phage-display technology.

C) Humanized or Chimeric Antibodies

The invention also provides anti-hTRT antibodies that are made chimeric,human-like or humanized, to reduce their potential antigenicity, withoutreducing their affinity for their target. Preparation of chimeric,human-like and humanized antibodies have been described in the art (see,e.g., U.S. Pat. Nos. 5,585,089 and 5,530,101; Queen, et al., 1989, Proc.Nat'l Acad. Sci. USA 86:10029; and Verhoeyan et al., 1988, Science239:1534; each of which is incorporated by reference in their entiretyand for all purposes). Humanized immunoglobulins have variable frameworkregions substantially from a human immunoglobulin (termed an acceptorimmunoglobulin) and complementarity determining regions substantiallyfrom a non-human (e.g., mouse) immunoglobulin (referred to as the donorimmunoglobulin). The constant region(s), if present, are alsosubstantially from a human immunoglobulin.

In some applications, such as administration to human patients, thehumanized (as well as human) anti-hTRT antibodies of the presentinvention offer several advantages over antibodies from murine or otherspecies: (1) the human immune system should not recognize the frameworkor constant region of the humanized antibody as foreign, and thereforethe antibody response against such an injected antibody should be lessthan against a totally foreign mouse antibody or a partially foreignchimeric antibody; (2) because the effector portion of the humanizedantibody is human, it may interact better with other parts of the humanimmune system; and (3) injected humanized antibodies have a half-lifeessentially equivalent to naturally occurring human antibodies, allowingsmaller and less frequent doses than antibodies of other species. Asimplicit from the foregoing, anti hTRT antibodies have application inthe treatment of disease, i.e., to target telomerase-positive cells.

D) Phage Display

The present invention also provides anti-hTRT antibodies (or bindingcompositions) produced by phage display methods (see, e.g., Dower etal., WO 91/17271 and McCafferty et al., WO 92/01047; and Vaughan et al.,1996, Nature Biotechnology, 14: 309; each of which is incorporated byreference in its entirety for all purposes). In these methods, librariesof phage are produced in which members display different antibodies ontheir outer surfaces. Antibodies are usually displayed as Fv or Fabfragments. Phage displaying antibodies with a desired specificity areselected by affinity enrichment to an hTRT polypeptide.

In a variation of the phage-display method, humanized antibodies havingthe binding specificity of a selected murine antibody can be produced.In this method, either the heavy or light chain variable region of theselected murine antibody is used as a starting material. If, forexample, a light chain variable region is selected as the startingmaterial, a phage library is constructed in which members display thesame light chain variable region (i.e., the murine starting material)and a different heavy chain variable region. The heavy chain variableregions are obtained from a library of rearranged human heavy chainvariable regions. A phage showing strong specific binding for the hTRTpolypeptide (e.g., at least 10⁸ and preferably at least 10⁹ M⁻¹) isselected. The human heavy chain variable region from this phage thenserves as a starting material for constructing a further phage library.In this library, each phage displays the same heavy chain variableregion (i.e., the region identified from the first display library) anda different light chain variable region. The light chain variableregions are obtained from a library of rearranged human variable lightchain regions. Again, phage showing strong specific binding areselected. These phage display the variable regions of completely humananti-hTRT antibodies. These antibodies usually have the same or similarepitope specificity as the murine starting material.

E) Hybrid Antibodies

The invention also provides hybrid antibodies that share the specificityof antibodies against an hTRT polypeptide but are also capable ofspecific binding to a second moiety. In such hybrid antibodies, oneheavy and light chain pair is usually from an anti-hTRT antibody and theother pair from an antibody raised against another epitope or protein.This results in the property of multi-functional valency, i.e., abilityto bind at least two different epitopes simultaneously, where at leastone epitope is the epitope to which the anti-complex antibody binds.Such hybrids can be formed by fusion of hybridomas producing therespective component antibodies, or by recombinant techniques. Suchhybrids can be used to carry a compound (i.e., drug) to atelomerase-positive cell (i.e., a cytotoxic agent is delivered to acancer cell).

Immunoglobulins of the present invention can also be fused to functionalregions from other genes (e.g., enzymes) to produce fusion proteins(e.g., immunotoxins) having useful properties.

F) Anti-Idiotypic Antibodies

Also useful are anti-idiotype antibodies which can be isolated by theabove procedures. Anti-idiotypic antibodies may be prepared by, forexample, immunization of an animal with the primary antibody (i.e.,anti-hTRT antibodies or hTRT-binding fragments thereof). For anti-hTRTantibodies, anti-idiotype antibodies whose binding to the primaryantibody is inhibited by an hTRT polypeptide or fragments thereof areselected. Because both the anti-idiotypic antibody and the hTRTpolypeptide or fragments thereof bind the primary immunoglobulin, theanti-idiotypic immunoglobulin can represent the “internal image” of anepitope and thus can substitute for the hTRT polypeptide in assays orcan be used to bind (i.e., inactivate) anti-hTRT antibodies, e.g., in apatient. Anti-idiotype antibodies can also interact with telomeraseassociated proteins. Administration of such antibodies can affecttelomerase function by titrating out or competing with hTRT in bindingto hTRT-associated proteins.

G) General

The antibodies of the invention may be of any isotype, e.g., IgM, IgD,IgG, IgA, and IgE, with IgG, IgA and IgM often preferred. Humanizedantibodies may comprise sequences from more than one class or isotype.

In another embodiment of the invention, fragments of the intactantibodies described above are provided. Typically, these fragments cancompete with the intact antibody from which they were derived forspecific binding to the hTRT polypeptide, and bind with an affinity ofat least 10⁷, 10⁸, 10⁹ M⁻¹, or 10¹⁰ M⁻¹. Antibody fragments includeseparate heavy chains, light chains, Fab, Fab′ F(ab′)₂, Fabc, and Fv.Fragments can be produced by enzymatic or chemical separation of intactimmunoglobulins. For example, a F(ab′)₂ fragment can be obtained from anIgG molecule by proteolytic digestion with pepsin at pH 3.0-3.5 usingstandard methods such as those described in Harlow and Lane, supra. Fabfragments may be obtained from F(ab′)₂ fragments by limited reduction,or from whole antibody by digestion with papain in the presence ofreducing agents (see generally, Paul, W., ed Fundamental Immunology 2ndRaven Press, N.Y., 1989, Ch. 7, incorporated by reference in itsentirety for all purposes). Fragments can also be produced byrecombinant DNA techniques. Segments of nucleic acids encoding selectedfragments are produced by digestion of full-length coding sequences withrestriction enzymes, or by de novo synthesis. Often fragments areexpressed in the form of phage-coat fusion proteins.

Many of the immunoglobulins described above can undergo non-criticalamino-acid substitutions, additions or deletions in both the variableand constant regions without loss of binding specificity or effectorfunctions, or intolerable reduction of binding affinity (i.e., belowabout 10⁷ M⁻¹). Usually, immunoglobulins incorporating such alterationsexhibit substantial sequence identity to a reference immunoglobulin fromwhich they were derived. A mutated immunoglobulin can be selected havingthe same specificity and increased affinity compared with a referenceimmunoglobulin from which it was derived. Phage-display technologyoffers useful techniques for selecting such immunoglobulins. See, e.g.,Dower et al., WO 91/17271 McCafferty et al., WO 92/01047; and Huse, WO92/06204.

The antibodies of the present invention can be used with or withoutmodification. Frequently, the antibodies will be labeled by joining,either covalently or non-covalently, a detectable label. As labeledbinding entities, the antibodies of the invention are particularlyuseful in diagnostic applications.

The anti-hTRT antibodies of the invention can be purified using wellknown methods. The whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention canbe purified using the methods and reagents of the present invention inaccordance with standard procedures of the art, including ammoniumsulfate precipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see generally Scopes, ProteinPurification: Principles and Practice 3rd Edition (Springer-Verlag,N.Y., 1994)). Substantially pure immunoglobulins of at least about 90 to95%, or even 98 to 99% or more homogeneity are preferred.

VI. Purification of Human Telomerase

The present invention provides isolated human telomerase ofunprecedented purity. In particular, the present invention provides:purified hTRT of recombinant or nonrecombinant origin; purified hTRT-hTRcomplexes (i.e., RNPs) of recombinant, nonrecombinant, or mixed origin,optionally comprising one or more telomerase-associated proteins;purified naturally occurring human telomerase; and the like. Moreover,the invention provides methods and reagents for partially, substantiallyor highly purifying the above-molecules and complexes, includingvariants, fusion proteins, naturally occurring proteins, and the like(collectively referred to as “hTRT and/or hTRT complexes”).

Prior to the present disclosure, attempts had been made to purify thetelomerase enzyme complex to homogeneity had met with limited success.The methods provided in the aforelisted applications providepurification of telomerase by approximately up to 60,000-fold or morecompared to crude cell extracts. The present invention provides hTRT andhTRT complexes of even greater purity, in part by virtue of the novelimmunoaffinity reagents (e.g., anti-hTRT antibodies) of the presentinvention, and/or the reagents, cells, and methods provided herein forrecombinant expression of hTRT. Recombinant expression of hTRT and hTRTcomplexes facilitates purification because the desired molecules can beproduced at much higher levels than found in most expressing cellsoccurring in nature, and/or because the recombinant hTRT molecule can bemodified (e.g., by fusion with an epitope tag) such that it may beeasily purified.

It will be recognized that naturally occurring telomerase can bepurified from any telomerase-positive cell, and recombinant hTRT andhTRT complexes can be expressed and purified, inter alia, using any ofthe in vitro, in vivo, ex vivo, or plant or animal expression systemsdisclosed supra, or others/systems known in the art.

In one embodiment, the hTRT, telomerase and other compositions of theinvention are purified using an immunoaffinity step, alone or incombination with other purification steps. Typically, an immobilized orimmobilizable anti-hTRT antibody, as provided by the present invention,is contacted with a sample, such as a cell lysate, that contains thedesired hTRT or hTRT-containing complex under conditions in whichanti-hTRT antibody binds the hTRT antigen. After removal of the unboundcomponents of the sample by methods well known in the art, the hTRTcomposition may be eluted, if desired, from the antibody, insubstantially pure form. In one embodiment, immunoaffinitychromatography methods well known in the art are used (see, e.g., Harlowand Lane, supra; and Ausubel, supra; Hermansan et al., 1992, ImmobilizedAffinity Ligand Techniques (Academic Press, San Diego)) in accordancewith the methods of the invention. In another illustrative embodiment,immunoprecipitation of anti-hTRT-immunoglobulin-hTRT complexes iscarried out using immobilized Protein A. Numerous variations andalternative imnmunoaffinity purification protocols suitable for use inaccordance with the methods and reagents of the invention are well-knownto those of skill.

In another embodiment, recombinant hTRT proteins can, as a consequenceof their high level of expression, be purified using routine proteinpurification methods, such as ammonium sulfate precipitation, affinitycolumns (e.g., immunoaffinity), size-exclusion, anion and cationexchange chromatography, gel electrophoresis and the like (see,generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982)and Deutscher, Methods in Enzymology Vol. 182: Guide to ProteinPurification, Academic Press, Inc. N.Y. (1990)) instead of, or inaddition to, immunoaffinity methods. Cation exchange methods can beparticularly useful due to the basic pI of the hTRT protein. Forexample, immobilized phosphate may be used as a cation exchangefunctional group (e.g., P-11 Phosphocellulose, Whatman catalog #4071 orCellulose Phosphate, Sigma catalog #C 3145). Immobilized phosphate hastwo advantageous features for hTRT purification—it is a cation exchangeresin, and it shows physical resemblance to the phosphate backbone ofnucleic acid. This can allow for affinity chromatography because hTRTbinds hTR and telomeric DNA. Other non-specific and specific nucleicacid affinity chromatography methods are also useful for purification(e.g., Alberts et al., 1971, Methods Enzymol. 21:198; Arnt-Jovin et al.,1975, Eur. J. Biochem. 54:411; Pharmacia catalog #27-5575-02). Furtherexploitation of this binding function of hTRT could include the use ofspecific nucleic acid (e.g., telomerase primer or hTR) affinitychromatography for purification (Chodosh et al., 1986, Mol. Cell. Biol.6:4723; Wu et al., 1987, Science 238:1247; Kadonaga, 1991, MethodsEnzymol. 208:10); immobilized Cibricon Blue Dye, which shows physicalresemblance to nucleotides, is another useful resin for hTRTpurification (Pharmacia catalog #17-0948-01 or Sigma catalog #C 1285),due to hTRT binding of nucleotides (e.g., as substrates for DNAsynthesis).

In one embodiment, hTRT proteins are isolated directly from an in vitroor in vivo expression system in which other telomerase components arenot coexpressed. It will be recognized that isolated hTRT protein mayalso be readily obtained from purified human telomerase or hTRTcomplexes, for example, by disrupting the telomerase RNP (e.g., byexposure to a mild or other denaturant) and separating the RNPcomponents (e.g., by routine means such as chromatography orimmunoaffinity chromatography).

Telomerase purification may be monitored using a telomerase activityassay (e.g., the TRAP assay, conventional assay, or primer-bindingassay), by measuring the enrichment of hTRT (e.g., by ELISA), bymeasuring the enrichment of hTR, or other methods known in the art.

The purified human telomerase, hTRT proteins, and hTRT complexesprovided by the present invention are, in one embodiment, highlypurified (i.e., at least about 90% homogeneous, more often at leastabout 95% homogeneous). Homogeneity can be determined by standard meanssuch as SDS-polyacrylamide gel electrophoresis and other means known inthe art (see, e.g., Ausubel et al, supra). It will be understood that,although highly purified human telomerase, hTRT protein, or hTRTcomplexes are sometimes desired, substantially purified (e.g., at leastabout 75% homogeneous) or partially purified (e.g., at least about 20%homogeneous) human telomerase, hTRT protein, or hTRT complexes areuseful in many applications, and are also provided by the presentinvention. For example, partially purified telomerase is useful forscreening test compounds for telomerase modulatory activity, and otheruses (see, infra and supra; see U.S. Pat. No. 5,645,986).

VII. Treatment of Telomerase-Related Disease

A) Introduction

The present invention provides hTRT polynucleotides, polypeptides, andantibodies useful for the treatment of human diseases and diseaseconditions. The recombinant and synthetic hTRT gene products (proteinand mRNA) of the invention can be used to create or elevate telomeraseactivity in a cell, as well as to inhibit telomerase activity in cellsin which it is not desired. Thus, inhibiting, activating or otherwisealtering a telomerase activity (e.g., telomerase catalytic activity,fidelity, processivity, telomere binding, etc.) in a cell can be used tochange the proliferative capacity of the cell. For example, reduction oftelomerase activity in an immortal cell, such as a malignant tumor cell,can render the cell mortal. Conversely, increasing the telomeraseactivity in a mortal cell (e.g., most human somatic cells) can increasethe proliferative capacity of the cell. For example, expression of hTRTprotein in dermal fibroblasts, thereby increasing telomere length, willresult in increased fibroblast proliferative capacity; such expressioncan slow or reverse the age-dependent slowing of wound closure (see,e.g., West, 1994, Arch. Derm. 130:87).

Thus, in one aspect, the present invention provides reagents and methodsuseful for treating diseases and conditions characterized by thepresence, absence, or amount of human telomerase activity in a cell andthat are susceptible to treatment using the compositions and methodsdisclosed herein. These diseases include, as described more fully below,cancers, other diseases of cell proliferation (particularly diseases ofaging), immunological disorders, infertility (or fertility), and others.

B) Treatment of Cancer

The present invention provides methods and compositions for reducingtelomerase activity in tumor cells and for treating cancer. Compositionsinclude antisense oligonucleotides, peptides, gene therapy vectorsencoding antisense oligonucleotides or activity altering proteins, andanti-hTRT antibodies. Cancer cells (e.g., malignant tumor cells) thatexpress telomerase activity (telomerase-positive cells) can bemortalized by decreasing or inhibiting the endogenous telomeraseactivity. Moreover, because telomerase levels correlate with diseasecharacteristics such as metastatic potential (e.g., U.S. Pat. Nos.5,639,613; 5,648,215; 5,489,508; Pandita et al., 1996, Proc. Am. Ass.Cancer Res. 37:559), any reduction in telomerase activity could reducethe aggressive nature of a cancer to a more manageable disease state(increasing the efficacy of traditional interventions).

The invention provides compositions and methods useful for treatment ofcancers of any of a wide variety of types, including solid tumors andleukemias. Types of cancer that may be treated include (but are notlimited to): adenocarcinoma of the breast, prostate, and colon; allforms of bronchogenic carcinoma of the lung; myeloid; melanoma;hepatoma; neuroblastoma; papilloma; apudoma; choristoma; branchioma;malignant carcinoid syndrome; carcinoid heart disease; carcinoma (e.g.,Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor,in situ, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell,papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, andtransitional cell), histiocytic disorders; leukemia (e.g., B-cell,mixed-cell, null-cell, T-cell, T-cell chronic, HTLV-II-associated,lyphocytic acute, lymphocytic chronic, mast-cell, and myeloid);histiocytosis malignant; Hodgkin's disease; immunoproliferative small;non-Hodgkin's lymphoma; plasmacytoma; reticuloendotheliosis; melanoma;chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giantcell tumors; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma;myxosarcoma; osteoma; osteosarcoma; Ewing's sarcoma; synovioma;adenofibroma; adenolymphoma; carcinosarcoma; chordoma;craniopharyngioma; dysgerminoma; hamartoma; mesenchymoma; mesonephroma;myosarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymoma;trophoblastic tumor; adenocarcinoma; adenoma; cholangioma;cholesteatoma; cylindroma; cystadenocarcinoma; cystadenoma; granulosacell tumor; gynandroblastoma; hepatoma; hidradenoma; islet cell tumor;leydig cell tumor; papilloma; sertoli cell tumor; theca cell tumor;leiomyoma; leiomyosarcoma; myoblastoma; myoma; myosarcoma; rhabdomyoma;rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma; medulloblastoma;meningioma; neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma;neuroma; paraganglioma; paraganglioma nonchromaffin; angiokeratoma;angiolymphoid hyperplasia with eosinophilia; angioma sclerosing;angiomatosis; glomangioma; hemangioendothelioma; hemangioma;hemangiopericytoma; hemangiosarcoma; lymphangioma; lymphangiomyoma;lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma;cystosarcoma phyllodes; fibrosarcoma; hemangiosarcoma; leiomyosarcoma;leukosarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma;ovarian carcinoma; rhabdomyosarcoma; sarcoma (e.g., Ewing's,experimental, Kaposi's, and mast-cell); neoplasms (e.g., bone, breast,digestive system, colorectal, liver, pancreatic, pituitary, testicular,orbital, head and neck, central nervous system, acoustic, pelvic,respiratory tract, and urogenital); neurofibromatosis, and cervicaldysplasia). The invention provides compositions and methods useful fortreatment of other conditions in which cells have become immortalized orhyperproliferative, e.g., by disregulation (e.g., abnormally highexpression) of hTRT, telomerase enzyme, or telomerase activity.

The present invention further provides compositions and methods forprevention of cancers, including anti-hTRT vaccines, gene therapyvectors that prevent telomerase activation, and gene therapy vectorsthat result in specific death of telomerase-positive cells. In a relatedaspect, the gene replacement therapy methods described below may be usedfor “treating” a genetic predilection for cancers.

C) Treatment of Other Conditions

The present invention also provides compositions and methods useful fortreatment of diseases and disease conditions (in addition to cancers)characterized by under- or over-expression of telomerase or hTRT geneproducts. Examples include: diseases of cell proliferation, diseasesresulting from cell senescence (particularly diseases of aging),immunological disorders, infertility, diseases of immune dysfunction,and others.

Certain diseases of aging are characterized by cellsenescence-associated changes due to reduced telomere length (comparedto younger cells), resulting from the absence (or much lower levels) oftelomerase activity in the cell. Decreased telomere length and decreasedreplicative capacity contribute to diseases such as those describedbelow. Telomerase activity and telomere length can be increased by, forexample, increasing levels of hTRT gene products (protein and mRNA) inthe cell. A partial listing of conditions associated with cellularsenescence in which hTRT expression can be therapeutic includesAlzheimer's disease, Parkinson's disease, Huntington's disease, andstroke; age-related diseases of the integument such as dermal atrophy,elastolysis and skin wrinkling, sebaceous gland hyperplasia, senilelentigo, graying of hair and hair loss, chronic skin ulcers, andage-related impairment of wound healing; degenerative joint disease;osteoporosis; age-related immune system impairment (e.g., involvingcells such as B and T lymphocytes, monocytes, neutrophils, eosinophils,basophils, NK cells and their respective progenitors); age-relateddiseases of the vascular system including atherosclerosis,calcification, thrombosis, and aneurysms; diabetes, muscle atrophy,respiratory diseases, diseases of the liver and GI tract, metabolicdiseases, endocrine diseases (e.g., disorders of the pituitary andadrenal gland), reproductive diseases, and age-related maculardegeneration. These diseases and conditions can be treated by increasingthe levels of hTRT gene products in the cell to increase telomerelength, thereby restoring or imparting greater replicative capacity tothe cell. Such methods can be carried out on cells cultured ex vivo orcells in vivo. In one embodiment, the cells are first treated toactivate telomerase and lengthen telomeres, and then treated toinactivate the hTRT gene and telomerase activity. In a preferredembodiment, telomerase activity is generated by a vector of theinvention in an embryonic germ or stem cell prior to or duringdifferentiation.

The present invention also provides methods and composition useful fortreating infertility. Human germline cells (e.g., spermatogonia cells,their progenitors or descendants) are capable of indefiniteproliferation and characterized by high telomerase activity. Abnormal ordiminished levels of hTRT gene products can result, for example, ininadequate or abnormal production of spermatozoa, leading to infertilityor disorders of reproduction. Accordingly, “telomerase-based”infertility can be treated using the methods and compositions describedherein to increase telomerase levels. Similarly, because inhibition oftelomerase may negatively impact spermatogenesis, oogenesis, and spermand egg viability, the telomerase inhibitory compositions of theinvention can have contraceptive effects when used to reduce hTRT geneproduct levels in germline cells.

Further, the invention provides methods and composition useful fordecreasing the proliferative potential of telomerase-positive cells suchas activated lymphocytes and hematopoietic stem cells by reducingtelomerase activity. Thus, the invention provide means for effectingimmunosuppression. Conversely, the methods and reagents of the inventionare useful for increasing telomerase activity and proliferativepotential in cells, such as stem cells, that express a low level oftelomerase or no telomerase prior to therapeutic intervention.

D) Modes of Intervention

As is clear from the foregoing discussion, modulation of the level oftelomerase or telomerase activity of a cell can have a profound effecton the proliferative potential of the cell, and so has great utility intreatment of disease. As is also clear, this modulation may be either adecrease in telomerase activity or an increase in activity. Thetelomerase modulatory molecules of the invention can act through anumber of mechanisms; some of these are described in this and thefollowing subsections to aid the practitioner in selecting therapeuticagents. However, applicants do not intend to be limited to anyparticular mechanism of action for the novel therapeutic compounds,compositions and methods described herein.

Telomerase activity may be decreased through any of several mechanismsor combinations of mechanisms. One mechanism is the reduction of hTRTgene expression to reduce telomerase activity. This reduction can be atthe level of transcription of the hTRT gene into mRNA, processing (e.g.,splicing), nuclear transport or stability of mRNA, translation of mRNAto produce hTRT protein, or stability and function of hTRT protein.Another mechanism is interference with one or more activities oftelomerase (e.g., the reverse transcriptase catalytic activity, or thehTR-binding activity) using inhibitory nucleic acids, polypeptides, orother agents (e.g., mimetics, small molecules, drugs and pro-drugs) thatcan be identified using the methods, or are provided by compositions,disclosed herein. Other mechanisms include sequestration of hTR and/ortelomerase associated proteins, and interference with the assembly ofthe telomerase RNP from its component subunits. In a related mechanism,an hTRT promoter sequence is operably linked to a gene encoding a toxinand introduced into a cell; if or when hTRT transcriptional activatorsare expressed or activated in the cell, the toxin will be expressed,resulting in specific cell killing.

A related method for reducing the proliferative capacity of a cellinvolves introducing an hTRT variant with low fidelity (i.e., one with ahigh, e.g., greater than 1%, error rate) such that aberrant telomericrepeats are synthesized. These aberrant repeats affect telomere proteinbinding and lead to chromosomal rearrangements and aberrations and/orlead to cell death.

Similarly, telomerase activity may be increased through any of severalmechanisms, or a combination of mechanisms. These include increasing theamount of hTRT in a cell. Usually this is carried out by introducing anhTRT polypeptide-encoding polynucleotide into the cell (e.g., arecombinantly produced polypeptide comprising an hTRT DNA sequenceoperably linked to a promoter, or a stable hTRT mRNA). Alternatively, acatalytically active hTRT polypeptide can itself be introduced into acell or tissue, e.g., by microinjection or other means known in the art.In other mechanisms, expression from the endogenous hTRT gene or thestability of hTRT gene products in the cell can be increased. Telomeraseactivity in a cell can also be increased by interfering with theinteraction of endogenous telomerase inhibitors and the telomerase RNP,or endogenous hTRT transcription repressors and the hTRT gene; byincreasing expression or activity of hTRT transcription activators; andother means apparent to those of skill upon review of this disclosure.

E) Intervention Agents

1) TRT Proteins & Peptides

In one embodiment, the invention provides telomerase modulatorypolypeptides (i.e., proteins, polypeptides, and peptides) that increaseor reduce telomerase activity which can be introduced into a target celldirectly (e.g., by injection, liposome-mediated fusion, application of ahydrogel to the tumor [e.g., melanoma] surface, fusion or attachment toherpes virus structural protein VP22, and other means described hereinand known in the art). In a second embodiment, telomerase modulatoryproteins and peptides of the invention are expressed in a cell byintroducing a nucleic acid (e.g., a DNA expression vector or mRNA)encoding the desired protein or peptide into the cell. Expression may beeither constitutive or inducible depending on the vector and choice ofpromoter (see discussion below). Messenger RNA preparations encodinghTRT are especially useful when only transient expression (e.g.,transient activation of telomerase) is desired. Methods for introductionand expression of nucleic acids into a cell are well known in the art(also, see elsewhere in this specification, e.g., sections onoligonucleotides, gene therapy methods).

In one aspect of the invention, a telomerase modulatory polypeptide thatincreases telomerase activity in a cell is provided. In one embodiment,the polypeptide is a catalytically active hTRT polypeptide capable ofdirecting the synthesis (in conjunction with an RNA template such ashTR) of human telomeric DNA. This activity can be measured, as discussedabove, e.g., using a telomerase activity assay such as a TRAP assay. Inone embodiment, the polypeptide is a full-length hTRT protein, having asequence of, or substantially identical to, the sequence of 1132residues of SEQ ID NO:2. In another embodiment, the polypeptide is avariant of the hTRT protein of SEQ ID NO:2, such as a fusionpolypeptide, derivatized polypeptide, truncated polypeptide,conservatively substituted-polypeptide, activity-modified polypeptide,or the like. A fusion or derivatized protein may include a targetingmoiety that increases the ability of the polypeptide to traverse a cellmembrane or causes the polypeptide to be delivered to a specified celltype (e.g., liver cells or tumor cells) preferentially or cellcompartment (e.g., nuclear compartment) preferentially. Examples oftargeting moieties include lipid tails, amino acid sequences such asantennapoedia peptide or a nuclear localization signal (NLS; e.g.,Xenopus nucleoplasmin Robbins et al., 1991, Cell 64:615). Naturallyoccurring hTRT protein (e.g., having a sequence of, or substantiallyidentical to, SEQ ID NO:2) acts in the cell nucleus. Thus, it is likelythat one or more subsequences of SEQ ID NO:2, such as residues 193-196(PRRR SEQ ID NO:541) and residues 235-240 (PKRPRR SEQ ID NO:542) act asa nuclear localization signal. The small regions are likely NLSs basedon the observation that many NLSs comprise a 4 residue pattern composedof basic amino acids (K or R), or composed of three basic amino acids (Kor R) and H or P; a pattern starting with P and followed within 3residues by a basic segment containing 3 K or R residues out of 4residues (see, e.g., Nakai et al., 1992, Genomics 14:897). Deletion ofone or both of these sequences and/or additional localization sequencesis expected to interfere with hTRT transport to the nucleus and/orincrease hTRT turnover, and is useful for preventing access oftelomerase to its nuclear substrates and decreasing proliferativepotential. Moreover, a variant hTRT polypeptide lacking NLS may assembleinto an RNP that will not be able to maintain telomere length, becausethe resulting enzyme cannot enter the nucleus.

The hTRT polypeptides of the invention will typically be associated inthe target cell with a telomerase RNA, such as hTR, especially when theyare used to increase telomerase activity in a cell. In one embodiment,an introduced hTRT polypeptide associates with an endogenous hTR to forma catalytically active RNP (e.g., an RNP comprising the hTR and afull-length polypeptide having a sequence of SEQ ID NO:2). The RNPso-formed may also associate with other, e.g., telomerase-associated,proteins. In other embodiments, telomerase RNP (containing hTRT protein,hTR and optionally other components) is introduced as a complex to thetarget cell.

In a related embodiment, an hTRT expression vector is introduced into acell (or progeny of a cell) into which a telomerase RNA (e.g., hTR)expression vector is simultaneously, subsequently or has been previouslyintroduced. In this embodiment, hTRT protein and telomerase RNA arecoexpressed in the cell and assemble to form a telomerase RNP. Apreferred telomerase RNA is hTR. An expression vector useful forexpression of hTR in a cell is described supra (see U.S. Pat. No.5,583,016). In yet another embodiment, the hTRT polypeptide and hTR RNA(or equivalent) are associated in vitro to form a complex, which is thenintroduced into the target cells, e.g., by liposome mediated transfer.

In another aspect, the invention provides hTRT polypeptides useful forreducing telomerase activity in a cell. As above, these “inhibitory”polypeptides can be introduced directly, or by expression of recombinantnucleic acids in the cell. It will be recognized that peptide mimeticsor polypeptides comprising nonstandard amino acids (i.e., other than the20 amino acids encoded by the genetic code or their normal derivatives)will typically be introduced directly.

In one embodiment, inhibition of telomerase activity results from thesequestration of a component required for accurate telomere elongation.Examples of such components are hTRT and hTR. Thus, administration of apolypeptide that binds hTR, but which does not have telomerase catalyticactivity, can reduce endogenous telomerase activity in the cell. In arelated embodiment, the hTRT polypeptide may bind a cell component otherthan hTR, such as one or more telomerase-associated proteins, therebyinterfering with telomerase activity in the cell.

In another embodiment, hTRT polypeptides of the invention interfere(e.g., by competition) with the interaction of endogenously expressedhTRT protein and another cellular component required for telomerasefunction, such as hTR, telomeric DNA, telomerase-associated proteins,telomere-associated proteins, telomeres, cell cycle control proteins,DNA repair enzymes, histone or non-histone chromosomal proteins, orothers.

In selecting molecules (e.g., polypeptides) of the invention that affectthe interaction of endogenously expressed hTRT protein and othercellular components, one may prefer molecules that include one or moreof the conserved motifs of the hTRT protein, as described herein. Theevolutionary conservation of these regions indicates the importantfunction in the proper functioning of human telomerase contributed bythese motifs, and the motifs are thus generally useful sites forchanging hTRT protein function to create variant hTRT proteins of theinvention. Thus, variant hTRT polypeptides having mutations in conservedmotifs will be particularly useful for some applications of theinvention.

In another embodiment, expression of the endogenous hTRT gene isrepressed by introduction into the cell of a large amount of hTRTpolypeptide (e.g., typically at least about 2-fold more than theendogenous level, more often at least about 10- to about 100-fold) whichacts via a feedback loop to inhibit transcription of the hTRT gene,processing of the hTRT pre-mRNA, translation of the hTRT mRNA, orassembly and transport of the telomerase RNP.

2) Oligonucleotides

a) Antisense Constructs

The invention provides methods and antisense oligonucleotide orpolynucleotide reagents which can be used to reduce expression of hTRTgene products in vitro or in vivo. Administration of the antisensereagents of the invention to a target cell results in reduced telomeraseactivity, and is particularly useful for treatment of diseasescharacterized by high telomerase activity (e.g., cancers). Withoutintending to be limited to any particular mechanism, it is believed thatantisense oligonucleotides bind to, and interfere with the translationof, the sense hTRT mRNA. Alternatively, the antisense molecule mayrender the hTRT mRNA susceptible to nuclease digestion, interfere withtranscription, interfere with processing, localization or otherwise withRNA precursors (“pre-mRNA”), repress transcription of mRNA from the hTRTgene, or act through some other mechanism. However, the particularmechanism by which the antisense molecule reduces hTRT expression is notcritical.

The antisense polynucleotides of the invention comprise an antisensesequence of at least 7 to 10 to typically 20 or more nucleotides thatspecifically hybridize to a sequence from mRNA encoding hTRT or mRNAtranscribed from the hTRT gene. More often, the antisense polynucleotideof the invention is from about 10 to about 50 nucleotides in length orfrom about 14 to about 35 nucleotides in length. In other embodiments,antisense polynucleotides are polynucleotides of less than about 100nucleotides or less than about 200 nucleotides. In general, theantisense polynucleotide should be long enough to form a stable duplexbut short enough, depending on the mode of delivery, to administer invivo, if desired. The minimum length of a polynucleotide required forspecific hybridization to a target sequence depends on several factors,such as G/C content, positioning of mismatched bases (if any), degree ofuniqueness of the sequence as compared to the population of targetpolynucleotides, and chemical nature of the polynucleotide (e.g.,methylphosphonate backbone, peptide nucleic acid, phosphorothioate),among other factors.

Generally, to assure specific hybridization, the antisense sequence issubstantially complementary to the target hTRT mRNA sequence In certainembodiments, the antisense sequence is exactly complementary to thetarget sequence. The antisense polynucleotides may also include,however, nucleotide substitutions, additions, deletions, transitions,transpositions, or modifications, or other nucleic acid sequences ornon-nucleic acid moieties so long as specific binding to the relevanttarget sequence corresponding to hTRT RNA or its gene is retained as afunctional property of the polynucleotide.

In one embodiment, the antisense sequence is complementary to relativelyaccessible sequences of the hTRT mRNA (e.g., relatively devoid ofsecondary structure). This can be determined by analyzing predicted RNAsecondary structures using, for example, the MFOLD program (GeneticsComputer Group, Madison Wis.) and testing in vitro or in vivo as isknown in the art. Examples of oligonucleotides that may be tested incells for antisense suppression of hTRT function are those capable ofhybridizing to (i.e., substantially complementary to) the followingpositions from SEQ ID NO:1: 40-60; 260-280; 500-520; 770-790; 885-905;1000-1020; 1300-1320; 1520-1540; 2110-2130; 2295-2315; 2450-2470;2670-2690; 3080-3110; 3140-3160; and 3690-3710. Another useful methodfor identifying effective antisense compositions uses combinatorialarrays of oligonucleotides (see, e.g., Milner et al., 1997, NatureBiotechnology 15:537).

The invention also provides an antisense polynucleotide that hassequences in addition to the antisense sequence (i.e., in addition toanti-hTRT-sense sequence). In this case, the antisense sequence iscontained within a polynucleotide of longer sequence. In anotherembodiment, the sequence of the polynucleotide consists essentially of,or is, the antisense sequence.

The antisense nucleic acids (DNA, RNA, modified, analogues, and thelike) can be made using any suitable method for producing a nucleicacid, such as the chemical synthesis and recombinant methods disclosedherein. In one embodiment, for example, antisense RNA molecules of theinvention may be prepared by de novo chemical synthesis or by cloning.For example, an antisense RNA that hybridizes to hTRT mRNA can be madeby inserting (ligating) an hTRT DNA sequence (e.g., SEQ ID NO:1, orfragment thereof) in reverse orientation operably linked to a promoterin a vector (e.g., plasmid). Provided that the promoter and, preferablytermination and polyadenylation signals, are properly positioned, thestrand of the inserted sequence corresponding to the noncoding strandwill be transcribed and act as an antisense oligonucleotide of theinvention.

The antisense oligonucleotides of the invention can be used to inhibittelomerase activity in cell-free extracts, cells, and animals, includingmammals and humans. For example, the phosphorothioate antisenseoligonucleotides:

A) 5″-GGCATCGCGGGGGTGGCCGGG SEQ ID NO:506 B) 5″-CAGCGGGGAGCGCGCGGCATCSEQ ID NO:521 C) 5″-CAGCACCTCGCGGTAGTGGCT SEQ ID NO:522 D)5″-GGACACCTGGCGGAAGGAGGG SEQ ID NO:507

can be used to inhibit telomerase activity. At 10 micromolarconcentration each oligonucleotide, mixtures of oligonucleotides A andB; A, B, C, and D; and A, C, and D inhibited telomerase activity in 293cells when treated once per day for seven days. Inhibition was alsoobserved when an antisense hTR molecule (5′-GCTCTAGAATGAAGGGTG-3′; 3′SEQ ID NO:543) was used in combination with oligonucleotides A, B, andC; A, B, and D; and A and C. Useful control oligonucleotides in suchexperiments include:

S1) 5″-GCGACGACTGACATTGGCCGG SEQ ID NO:544 S2) 5″-GGCTCGAAGTAGCACCGGTGCSEQ ID NO:545 S3) 5″-GTGGGAACAGGCCGATGTCCC SEQ ID NO:546

To determine the optimum antisense oligonucleotide of the invention forthe particular application of interest, one can perform a scan usingantisense oligonucleotide sets of the invention. One illustrative set isthe set of 30-mer oligonucleotides that span the hTRT mRNA and areoffset one from the next by fifteen nucleotides (i.e., ON1 correspondsto positions 1-30 and is TCCCACGTGCGCAGCAGGACGCAGCGCTGC (SEQ ID NO:547),ON2 corresponds to positions 16-45 and is GCCGGGGCCAGGGCTTCCCACGTGCGCAGC(SEQ ID NO:548), and ON3 corresponds to positions 31-60 and isGGCATCGCGGGGGTGGCCGGGGCCAGGGCT (SEQ ID NO:549), and so on to the end ofthe mRNA). Each member of this set can be tested for inhibitory activityas disclosed herein. Those oligonucleotides that show inhibitoryactivity under the conditions of interest then identify a region ofinterest, and other oligonucleotides of the invention corresponding tothe region of interest (i.e., 8-mers, 10-mers, 15-mers, and so on) canbe tested to identify the oligonucleotide with the preferred activityfor the application.

Exemplary antisense oligonucleotides include 5′-GGCATCGCGGGGGTGGCCGGGGCCAGGGCT-3′ (SEQ ID NO:722) (corresponding to nucleotidepositions 31-60 of hTRT); 5′-GCGCA GCGTGCCAGCAGGTGAACCAGCACG-3′ (SEQ IDNO:723) (corresponding to positions 496-525);5′-GCCCGTTCGCATCCCAGACGCCTTCGGGGT-3′ (SEQ ID NO:724) (corresponding topositions 631-660); and 5′-ACGCTATGGTTCCAGGCCCGTTCGCATCCC-3′ (SEQ IDNO:725) (corresponding to positions 646-675). When ACHN cells (NCI#503755) or 293 cells were treated for three days with 10 μM ofphosphorothioate oligonucleotides with any of the four sequences supra,inhibition of telomerase activity by about 50%-90% (compared to controluntreated cells) as measured by a TRAP assay, was observed.

For general methods relating to antisense polynucleotides, see AntisenseRNA and DNA, (1988), D. A. Melton, Ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.). See also, Dagle et al., 1991, Nucleic AcidsResearch, 19:1805. For a review of antisense therapy, see, e.g., Uhlmannet al., Chem. Reviews, 90:543-584 (1990).

b) Triplex Oligo- and Polynucleotides

The present invention provides oligo- and polynucleotides (e.g., DNA,RNA, PNA or the like) that bind to double-stranded or duplex hTRTnucleic acids (e.g., in a folded region of the hTRT RNA or in the hTRTgene), forming a triple helix-containing, or “triplex” nucleic acid.Triple helix formation results in inhibition of hTRT expression by, forexample, preventing transcription of the hTRT gene, thus reducing oreliminating telomerase activity in a cell. Without intending to be boundby any particular mechanism, it is believed that triple helix pairingcompromises the ability of the double helix to open sufficiently for thebinding of polymerases, transcription factors, or regulatory moleculesto occur.

Triplex oligo- and polynucleotides of the invention are constructedusing the base-pairing rules of triple helix formation (see, e.g., Chenget al., 1988, J. Biol. Chem. 263: 15110; Ferrin and Camerini-Otero,1991, Science 354:1494; Ramdas et al., 1989, J. Biol. Chem. 264:17395;Strobel et al., 1991, Science 254:1639; and Rigas et al., 1986, Proc.Natl. Acad. Sci. U.S.A. 83: 9591; each of which is incorporated hereinby reference) and the hTRT mRNA and/or gene sequence. Typically, thetriplex-forming oligonucleotides of the invention comprise a specificsequence of from about 10 to at least about 25 nucleotides or longer“complementary” to a specific sequence in the hTRT RNA or gene (i.e.,large enough to form a stable triple helix, but small enough, dependingon the mode of delivery, to administer in vivo, if desired). In thiscontext, “complementary” means able to form a stable triple helix. Inone embodiment, oligonucleotides are designed to bind specifically tothe regulatory regions of the hTRT gene (e.g., the hTRT 5′-flankingsequence, promoters, and enhancers) or to the transcription initiationsite, (e.g., between −10 and +10 from the transcription initiationsite). For a review of recent therapeutic advances using triplex DNA,see Gee et al., in Huber and Carr, 1994, Molecular and ImmunologicApproaches, Futura Publishing Co, Mt Kisco N.Y. and Rininsland et al.,1997, Proc. Natl. Acad. Sci. USA 94:5854, which are both incorporatedherein by reference.

c) Ribozymes

The present invention also provides ribozymes useful for inhibition oftelomerase activity. The ribozymes of the invention bind andspecifically cleave and inactivate hTRT mRNA. Useful ribozymes cancomprise 5′- and 3′-terminal sequences complementary to the hTRT mRNAand can be engineered by one of skill on the basis of the hTRT mRNAsequence disclosed herein (see PCT publication WO 93/23572, supra).Ribozymes of the invention include those having characteristics of groupI intron ribozymes (Cech, 1995, Biotechnology 13:323) and others ofhammerhead ribozymes (Edgington, 1992, Biotechnology 10:256).

Ribozymes of the invention include those having cleavage sites such asGUA, GUU) and GUC. Other optimum cleavage sites for ribozyme-mediatedinhibition of telomerase activity in accordance with the presentinvention include those described in PCT publications WO 94/02595 and WO93/23569, both incorporated herein by reference. Short RNAoligonucleotides between 15 and 20 ribonucleotides in lengthcorresponding to the region of the target hTRT gene containing thecleavage site can be evaluated for secondary structural features thatmay render the oligonucleotide more desirable. The suitability ofcleavage sites may also be evaluated by testing accessibility tohybridization with complementary oligonucleotides using ribonucleaseprotection assays, or by testing for in vitro ribozyme activity inaccordance with standard procedures known in the art.

As described by Hu et al., PCT publication WO 94/03596, incorporatedherein by reference, antisense and ribozyme functions can be combined ina single oligonucleotide. Moreover, ribozymes can comprise one or moremodified nucleotides or modified linkages between nucleotides, asdescribed above in conjunction with the description of illustrativeantisense oligonucleotides of the invention.

In one embodiment, the ribozymes of the invention are generated in vitroand introduced into a cell or patient. In another embodiment, genetherapy methods are used for expression of ribozymes in a target cell exvivo or in vivo.

d) Administration of Oligonucleotides

Typically, the therapeutic methods of the invention involve theadministration of an oligonucleotide that functions to inhibit orstimulate telomerase activity under in vivo physiological conditions,and is relatively stable under those conditions for a period of timesufficient for a therapeutic effect. As noted above, modified nucleicacids may be useful in imparting such stability, as well as fortargeting delivery of the oligonucleotide to the desired tissue, organ,or cell.

Oligo- and poly-nucleotides can be delivered directly as a drug in asuitable pharmaceutical formulation, or indirectly by means ofintroducing a nucleic acid into a cell, including liposomes,immunoliposomes, ballistics, direct uptake into cells, and the like asdescribed herein. For treatment of disease, the oligonucleotides of theinvention will be administered to a patient in a therapeuticallyeffective amount. A therapeutically effective amount is an amountsufficient to ameliorate the symptoms of the disease or modulatetelomerase activity in the target cell, e.g., as can be measured using aTRAP assay or other suitable assay of telomerase biological function.Methods useful for delivery of oligonucleotides for therapeutic purposesare described in U.S. Pat. No. 5,272,065, incorporated herein byreference. Other details of administration of pharmaceutically activecompounds are provided below. In another embodiment, oligo- andpoly-nucleotides can be delivered using gene therapy and recombinant DNAexpression plasmids of the invention.

3) Gene Therapy

Gene therapy refers to the introduction of an otherwise exogenouspolynucleotide which produces a medically useful phenotypic effect uponthe (typically) mammalian cell(s) into which it is transferred. In oneaspect, the present invention provides gene therapy methods andcompositions for treatment of telomerase-associated conditions. Inillustrative embodiments, gene therapy involves introducing into a cella vector that expresses an hTRT gene product (such as an hTRT proteinsubstantially similar to the hTRT polypeptide having a sequence ofSEQUENCE ID NO:2, e.g., to increase telomerase activity, or aninhibitory hTRT polypeptide to reduce activity), expresses a nucleicacid having an hTRT gene or mRNA sequence (such as an antisense RNA,e.g., to reduce telomerase activity), expresses a polypeptide orpolynucleotide that otherwise affects expression of hTRT gene products(e.g., a ribozyme directed to hTRT mRNA to reduce telomerase activity),or replaces or disrupts an endogenous hTRT sequence (e.g., genereplacement and “gene knockout,” respectively). Numerous otherembodiments will be evident to one of skill upon review of thedisclosure herein. In one embodiment, a vector encoding hTR is alsointroduced. In another embodiment, vectors encodingtelomerase-associated proteins are also introduced with or without avector for hTR.

Vectors useful in hTRT gene therapy can be viral or nonviral, andinclude those described supra in relation to the hTRT expression systemsof the invention. It will be understood by those of skill in the artthat gene therapy vectors may comprise promoters and other regulatory orprocessing sequences, such as are described in this disclosure. Usuallythe vector will comprise a promoter and, optionally, an enhancer(separate from any contained within the promoter sequences) that serveto drive transcription of an oligoribonucleotide, as well as otherregulatory elements that provide for episomal maintenance or chromosomalintegration and for high-level transcription, if desired. A plasmiduseful for gene therapy can comprise other functional elements, such asselectable markers, identification regions, and other sequences. Theadditional sequences can have roles in conferring stability both outsideand within a cell, targeting delivery of hTRT nucleotide sequences(sense or antisense) to a specified organ, tissue, or cell population,mediating entry into a cell, mediating entry into the nucleus of a celland/or mediating integration within nuclear DNA. For example,aptamer-like DNA structures, or other protein binding moieties sites canbe used to mediate binding of a vector to cell surface receptors or toserum proteins that bind to a receptor thereby increasing the efficiencyof DNA transfer into the cell. Other DNA sites and structures candirectly or indirectly bind to receptors in the nuclear membrane or toother proteins that go into the nucleus, thereby facilitating nuclearuptake of a vector. Other DNA sequences can directly or indirectlyaffect the efficiency of integration.

Suitable gene therapy vectors may, or may not, have an origin ofreplication. For example, it is useful to include an origin ofreplication in a vector for propagation of the vector prior toadministration to a patient. However, the origin of replication canoften be removed before administration if the vector is designed tointegrate into host chromosomal DNA or bind to host mRNA or DNA. In somesituations (e.g., tumor cells) it may not be necessary for the exogenousDNA to integrate stably into the transduced cell, because transientexpression may suffice to kill the tumor cells.

As noted, the present invention also provides methods and reagents forgene replacement therapy (i.e., replacement by homologous recombinationof an endogenous hTRT gene with a recombinant gene). Vectorsspecifically designed for integration by homologous recombination may beused. Important factors for optimizing homologous recombination includethe degree of sequence identity and length of homology to chromosomalsequences. The specific sequence mediating homologous recombination isalso important, because integration occurs much more easily intranscriptionally active DNA. Methods and materials for constructinghomologous targeting constructs are described by e.g., Mansour et al.,1988, Nature 336: 348; Bradley et al., 1992, Bio/Technology 10: 534. Seealso, U.S. Pat. Nos. 5,627,059; 5,487,992; 5,631,153; and 5,464,764. Inone embodiment, gene replacement therapy involves altering or replacingall or a portion of the regulatory sequences controlling expression ofthe hTRT gene that is to be regulated. For example, the hTRT promotersequences (e.g., such as are found in SEQ ID NO:6) may be disrupted (todecrease hTRT expression or to abolish a transcriptional control site)or an exogenous promoter (e.g., to increase hTRT expression)substituted.

The invention also provides methods and reagents for hTRT “geneknockout” (i.e., deletion or disruption by homologous recombination ofan endogenous hTRT gene using a recombinantly produced vector). In geneknockout, the targeted sequences can be regulatory sequences (e.g., thehTRT promoter), or RNA or protein coding sequences. The use ofhomologous recombination to alter expression of endogenous genes isdescribed in detail in U.S. Pat. No. 5,272,071 (and the U.S. Patentscited supra), WO 91/09955, WO 93/09222, WO 96/29411, WO 95/31560, and WO91/12650. See also, Moynahan et al., 1996, Hum. Mol. Genet. 5:875.

The invention further provides methods for specifically killingtelomerase-positive cells, or preventing transformation of telomerasenegative cells to a telomerase positive state, using the hTRT genepromoter to regulate expression of a protein toxic to the cell. As shownin Example 14, an hTRT promoter sequence may be operably linked to areporter gene such that activation of the promoter results in expressionof the protein encoded by the reporter gene. If, instead of a reporterprotein, the encoded protein is toxic to the cell, activation of thepromoter leads to cell morbidity or death. In one embodiment of thepresent invention, a vector comprising an hTRT promoter operably linkedto a gene encoding a toxic protein is introduced into cells, such ashuman cells, e.g., cells in a human patient, resulting in cell death ofcells in which hTRT promoter activating factors are expressed, such ascancer cells. In a related embodiment, the encoded protein is not itselftoxic to a cell, but encodes an activity that renders the cell sensitiveto an otherwise nontoxic drug. For example, tumors can be treated byintroducing an hTRT-promoter-Herpes thymidine kinase (TK) gene fusionconstruct into tumor cells, and administering gancyclovir or theequivalent (see, e.g., Moolton and Wells, 1990, J. Nat'l Canc. Inst.82:297). The art knows of numerous other suitable toxic or potentiallytoxic proteins and systems (using promoter sequences other that hTRT)that may be modified and applied in accordance with the presentinvention by one of skill in the art upon review of this disclosure.

Gene therapy vectors may be introduced into cells or tissues in vivo, invitro or ex vivo. For ex vivo therapy, vectors may be introduced intocells, e.g., stem cells, taken from the patient and clonally propagatedfor autologous transplant back into the same patient (see, e.g., U.S.Pat. Nos. 5,399,493 and 5,437,994, the disclosures of which are hereinincorporated by reference). Cells that can be targeted for hTRT genetherapy aimed at increasing the telomerase activity of a target cellinclude, but are not limited to, embryonic stem or germ cells,particularly primate or human cells, as noted supra, hematopoietic stemcells (AIDS and post-chemotherapy), vascular endothelial cells (cardiacand cerebral vascular disease), skin fibroblasts and basal skinkeratinocytes (wound healing and burns), chondrocytes (arthritis), brainastrocytes and microglial cells (Alzheimer's Disease), osteoblasts(osteoporosis), retinal cells (eye diseases), and pancreatic islet cells(Type I diabetes) and any of the cells listed in Table 3, infra, as wellas any other cell types known to divide.

In one embodiment of the invention, an inducible promoter operablylinked to a TRT, such as HTRT, coding sequence (or variant) is used tomodulate the proliferative capacity of cells in vivo or in vitro. In aparticular embodiment, for example, insulin-producing pancreatic cellstransfected with an hTRT expression vector under the control of aninducible promoter are introduced into a patient. The proliferativecapacity of the cells can then be controlled by administration to thepatient of the promoter activating agent (e.g., tetracycline) to enablethe cells to multiply more than otherwise would have been possible. Cellproliferation can then be terminated, continued, or reinitiated asdesired by the treating physician.

4) Vaccines and Antibodies

Immuogenic peptides or polypeptides having an hTRT sequence can be usedto elicit an anti-hTRT immune response in a patient (i.e., act as avaccine). Exemplary immunogenic hTRT peptides and polypeptides aredescribed infra in Examples 6 and 8. An immune response can also beraised by delivery of plasmid vectors encoding the polypeptide ofinterest (i.e., administration of “naked DNA”). The nucleic acids ofinterest can be delivered by injection, liposomes, or other means ofadministration. In one embodiment, immunization modes that elicit in thesubject a Class I MHC restricted cytotoxic lymphocyte response againsttelomerase expressing cells are chosen. Once immunized, the individualor animal will elicit a heightened immune response against cellsexpressing high levels of telomerase (e.g., malignant cells).

Anti-hTRT antibodies, e.g., murine, human, or humanized monoclonalantibodies may also be administered to a patient (e.g., passiveimmunization) to effect an immune response against telomerase-expressingcells.

F) Pharmaceutical Compositions

In related aspects, the invention provides pharmaceutical compositionsthat comprise hTRT oligo- and poly-nucleotides, polypeptides, andantibodies, agonists, antagonists, or inhibitors, alone or incombination with at least one other agent, such as a stabilizingcompound, diluent, carrier, or another active ingredient or agent.

The therapeutic agents of the invention may be administered in anysterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Any of thesemolecules can be administered to a patient alone, or in combination withother agents, drugs or hormones, in pharmaceutical compositions where itis mixed with suitable excipient(s), adjuvants, and/or pharmaceuticallyacceptable carriers. In one embodiment of the present invention, thepharmaceutically acceptable carrier is pharmaceutically inert.

Administration of pharmaceutical compositions is accomplished orally orparenterally. Methods of parenteral delivery include topical,intra-arterial (e.g., directly to the tumor), intramuscular,subcutaneous, intramedullary, intrathecal, intraventricular,intravenous, intraperitoneal, or intranasal administration. In additionto the active ingredients, these pharmaceutical compositions may containsuitable pharmaceutically acceptable carriers comprising excipients andother compounds that facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Maack Publishing Co,Easton Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, etc., suitablefor ingestion by the patient. See PCT publication WO 93/23572.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable additional compounds, if desired, to obtaintablets or dragee cores. Suitable excipients are carbohydrate or proteinfillers include, but are not limited to sugars, including lactose,sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato,or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic and tragacanth; as well as proteins such asgelatin and collagen. If desired, disintegrating or solubilizing agentsmay be added, such as the cross-linked polyvinyl pyrrolidone, agar,alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage).

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active compoundsmay be prepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner similar to that known in the art (e.g., bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses).

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions comprising a compound of the inventionformulated in a acceptable carrier have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of human telomerase proteins andnucleic acids, such labeling would include amount, frequency and methodof administration.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. “Therapeuticallyeffective amount” or “pharmacologically effective amount” are wellrecognized phrases and refer to that amount of an agent effective toproduce the intended pharmacological result. Thus, a therapeuticallyeffective amount is an amount sufficient to ameliorate the symptoms ofthe disease being treated. One useful assay in ascertaining an effectiveamount for a given application (e.g., a therapeutically effectiveamount) is measuring the effect on telomerase activity in a target cell.The amount actually administered will be dependent upon the individualto which treatment is to be applied, and will preferably be an optimizedamount such that the desired effect is achieved without significantside-effects. The determination of a therapeutically effective dose iswell within the capability of those skilled in the art.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in any appropriate animalmodel. The animal model is also used to achieve a desirableconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans.

A therapeutically effective amount refers to that amount of protein,polypeptide, peptide, antibody, oligo- or polynucleotide, agonist orantagonists which ameliorates the symptoms or condition. Therapeuticefficacy and toxicity of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals(e.g., ED₅₀, the dose therapeutically effective in 50% of thepopulation; and LD₅₀, the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, ED₅₀/LD₅₀. Pharmaceuticalcompositions which exhibit large therapeutic indices are preferred. Thedata obtained from cell culture assays and animal studies is used informulating a range of dosage for human use. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage varieswithin this range depending upon the dosage form employed, sensitivityof the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state (e.g., tumor size and location; age,weight and gender of the patient; diet, time and frequency ofadministration, drug combination(s), reaction sensitivities andtolerance/response to therapy). Long acting pharmaceutical compositionsmight be administered every 3 to 4 days, every week, or once every twoweeks depending on half-life and clearance rate of the particularformulation. Guidance as to particular dosages and methods of deliveryis provided in the literature (see, U.S. Pat. Nos. 4,657,760; 5,206,344;and 5,225,212, herein incorporated by reference). Those skilled in theart will typically employ different formulations for nucleotides thanfor proteins or their inhibitors. Similarly, delivery of polynucleotidesor polypeptides can be specific to particular cells, conditions,locations, and the like.

VIII. Increasing Proliferative Capacity and Production of ImmortalizedCells, Cell Lines, and Animals

As discussed above, most vertebrate cells senesce after a finite numberof divisions in culture (e.g., 50 to 100 divisions). Certain variantcells, however, are able to divide indefinitely in culture (e.g., HeLacells, 293 cells) and, for this reason, are useful for research andindustrial applications. Usually these immortal cell lines are derivedfrom spontaneously arising tumors, or by transformation by exposure toradiation or a tumor-inducing virus or chemical. Unfortunately, alimited selection of cell lines, especially human cell linesrepresenting differentiated cell function, is available. Moreover, theimmortal cell lines presently available are characterized by chromosomalabnormalities (e.g., aneuploidy, gene rearrangements, or mutations).Further, many long-established cell lines are relativelyundifferentiated (e.g., they do not produce highly specialized productsof the sort that uniquely characterize particular tissues or organs).Thus, there is a need for new methods of generating immortal cells,especially human cells. As used herein, the term “immortalized cells” isnot limited to cells that proliferate indefinitely, but may also includecells with increased proliferative capacity compared to similarwild-type cells. Depending on the cell type, increased proliferativecapacity may mean proliferation for at least about 100, about 150, about200, or about 400 or more generations, or for at least about 6, about12, about 18, about 24 or about 36 or more months in in vitro culture.One use for immortalized cells is in production of natural proteins andrecombinant proteins (e.g., therapeutic polypeptides such aserytbropoietin, human growth hormone, insulin, and the like), orantibodies, for which a stable, genetically normal cell line ispreferred. For production of some recombinant proteins, specialized celltypes may also be preferred (e.g., pancreatic cells for the productionof human insulin). Another use for immortalized cells or even mortalcells with increased proliferative capacity (relative to unmodifiedcells) is for introduction into a patient for gene therapy, or forreplacement of diseased or damaged cells or tissue. For example,autologous immune cells containing or expressing a, e.g., recombinanthTRT gene or polypeptide of the invention can be used for cellreplacement in a patient after aggressive cancer therapy, e.g., wholebody irradiation. Another use for immortalized cells is for ex vivoproduction of “artificial” tissues or organs (e.g., skin) fortherapeutic use. Another use for such cells is for screening orvalidation of drugs, such as telomerase-inhibiting drugs, or for use inproduction of vaccines or biological reagents. Additional uses of thecells of the invention will be apparent to those of skill.

The immortalized cells and cell lines, as well as those of merelyincreased replicative capacity, of the invention are made by increasingtelomerase activity in the cell. Any method disclosed herein forincreasing telomerase activity can be used. Thus, in one embodiment,cells are immortalized by increasing the amount of an hTRT polypeptidein the cell. In one embodiment, hTRT levels are increased by introducingan hTRT expression vector into the cell (with stable transfectionsometimes preferred). As discussed above, the hTRT coding sequence isusually operably linked to a promoter, which may be inducible orconstitutively active in the cell.

In one embodiment, a polynucleotide comprising a sequence encoding apolypeptide of SEQ ID NO:2, which sequence is operably linked to apromoter (e.g., a constitutively expressed promoter, e.g., a sequence ofSEQ ID NO:6, is introduced into the cell. In one embodiment thepolynucleotide comprises a sequence of SEQ ID NO:1. Preferably thepolynucleotide includes polyadenylation and termination signals. Inother embodiments, additional elements such as enhancers or othersdiscussed supra are included. In an alternative embodiment, thepolynucleotide does not include a promoter sequence, such sequence beingprovided by the target cell endogenous genome following integration(e.g., recombination, e.g., homologous recombination) of the introducedpolynucleotide. The polynucleotide may be introduced into the targetcell by any method, including any method disclosed herein, such aslipofection, electroporation, virosomes, liposomes, immnunoliposomes,polycation:nucleic acid conjugates, naked DNA).

Using the methods of the invention, any vertebrate cell can be caused tohave an increased proliferative capacity or even be immortalized andsustained indefinitely in culture. In one embodiment the cells aremammalian, with human cells preferred for many applications. Examples ofhuman cells that can be immortalized include those listed in Table 3.

It will be recognized that the “diagnostic” assays of the inventiondescribed infra may be used to identify and characterize theimmortalized cells of the invention.

TABLE 3 HUMAN CELLS IN WHICH hTRT EXPRESSION MAY BE INCREASEDKeratinizing Epithelial Cells keratinocyte of epidermis (differentiatingepidermal cell) basal cell of epidermis (stem cell) keratinocyte offingernails and toenails basal cell of nail bed (stem cell) hair shaftcells medullary, cortical, cuticular; hair-root sheath cells, cuticular,of Huxley's layer, of Henle's layer external; hair matrix cell (stemcell) Cells of Wet Stratified Barrier Epithelia surface epithelial cellof stratified squamous epithelium of tongue, oral cavity, esophagus,anal canal, distal urethra, vagina basal cell of these epithelia (stemcell) cell of external corneal epithelium cell of urinary epithelium(lining bladder and urinary ducts) Epithelial Cells Specialized forExocrine Secretion cells of salivary gland mucous cell (secretion richin polysaccharide) serous cell (secretion rich in glycoprotein enzymes)cell of von Ebner's gland in tongue (secretion to wash over taste buds)cell of mammary gland, secreting milk cell of lacrimal gland, secretingtears cell of ceruminous gland of ear, secreting wax cell of eccrinesweat gland, secreting glycoproteins (dark cell) cell of eccrine sweatgland, secreting small molecules (clear cell) cell of apocrine sweatgland (odoriferous secretion, sex- hormone sensitive) cell of gland ofMoll in eyelid (specialized sweat gland) cell of sebaceous gland,secreting lipid-rich sebum cell of Bowman's gland in nose (secretion towash over olfactory epithelium) cell of Brunner's gland in duodenum,secreting alkaline solution of mucus and enzymes cell of seminalvesicle, secreting components of seminal fluid, including fructose (asfuel for swimming sperm) cell of prostate gland, secreting othercomponents of seminal fluid cell of bulbourethral gland, secreting mucuscell of Bartholin's gland, secreting vaginal lubricant cell of gland ofLittré, secreting mucus cell of endometrium of uterus, secreting mainlycarbohydrates isolated goblet cell of respiratory and digestive tracts,secreting mucus mucous cell of lining of stomach zymogenic cell ofgastric gland, secreting pepsinogen oxyntic cell of gastric gland,secreting HCl acinar cell of pancreas, secreting digestive enzymes andbicarbonate Paneth cell of small intestine, secreting lysozyme type IIpneumocyte of lung, secreting surfactant Clara cell of lung Cellsspecialized for Secretion of Hormones cells of anterior pituitary,secreting growth hormone, follicle-stimulating hormone, luteinizinghormone, prolactin, adrenocorticotropic hormone, and thyroid-stimulatinghormone, cell of intermediate pituitary, secretingmelanocyte-stimulating hormone cells of posterior pituitary, secretingoxytocin, vasopressin cells of gut, secreting serotonin, endorphin,somatostatin, gastrin, secretin, cholecystokinin, insulin and glucagoncells of thyroid gland, secreting thyroid hormone, calcitonin cells ofparathyroid gland, secreting parathyroid hormone, oxyphil cell cells ofadrenal gland, secreting epinephrine, norepinephrine, and steroidhormones; mineralocorticoids glucocorticoids cells of gonads, secretingtestosterone (Leydig cell of testis) estrogen (theca interna cell ofovarian follicle) progesterone (corpus luteum cell of ruptured ovarianfollicle) cells of juxtaglomerular apparatus of kidney juxtaglomerularcell (secreting renin) macula densa cell peripolar cell mesangial cellEpithelial Absorptive Cells in Gut, Exocrine Glands, and UrogenitalTract brush border cell of intestine (with microvilli) striated ductcell of exocrine glands gall bladder epithelial cell brush border cellof proximal tubule of kidney distal tubule cell of kidney nonciliatedcell of ductulus efferens epididymal principal cell epididymal basalcell Cells Specialized for Metabolism and Storage hepatocyte (livercell) fat cells white fat brown fat lipocyte of liver Epithelial CellsServing Primarily a Barrier Function, Lining the Lung, Gut, ExocrineGlands, and Urogenital Tract type I pneumocyte (lining air space oflung) pancreatic duct cell (centroacinar cell) nonstriated duct cell ofsweat gland, salivary gland, mammary gland parietal cell of kidneyglomerulus podocyte of kidney glomerulus cell of thin segment of loop ofHenle (in kidney) collecting duct cell (in kidney) duct cell of seminalvesicle, prostate gland Epithelial Cells Lining Closed Internal BodyCavities vascular endothelial cells of blood vessels and lymphaticsfenestrated continuous splenic synovial cell (lining joint cavities,secreting largely hyaluronic acid) serosal cell (lining peritoneal,pleural, and pericardial cavities) squamous cell lining perilymphaticspace of ear cells lining endolymphatic space of ear squamous cellcolumnar cells of endolymphatic sac with microvilli without microvilli“dark” cell vestibular membrane cell (resembling choroid plexus cell)stria vascularis basal cell stria vascularis marginal cell cell ofClaudius cell of Boettcher choroid plexus cell (secreting cerebrospinalfluid) squamous cell of pia-arachnoid cells of ciliary epithelium of eyepigmented nonpigmented corneal “endothelial” cell Ciliated Cells withPropulsive Function of respiratory tract of oviduct and of endometriumof uterus (in female) of rete testis and ductulus efferens (in male) ofcentral nervous system (ependymal cell lining brain cavities) CellsSpecialized for Secretion of Extracellular Matrix epithelial: ameloblast(secreting enamel of tooth) planum semilunatum cell of vestibularapparatus of ear (secreting proteoglycan) interdental cell of organ ofCorti (secreting tectorial “membrane” covering hair cells of organ ofCorti) nonepithelial (connective tissue) fibroblasts (various-of looseconnective tissue, of cornea, of tendon, of reticular tissue of bonemarrow, etc.) pericyte of blood capillary nucleus pulposus cell ofintervertebral disc cementoblast/cementocyte (secreting bonelikecementum of root of tooth) odontoblast/odontocyte (secreting dentin oftooth) chondrocytes of hyaline cartilage, of fibrocartilage, of elasticcartilage osteoblast/osteocyte osteoprogenitor cell (stem cell ofosteoblasts) hyalocyte of vitreous body of eye stellate cell ofperilymphatic space of ear Contractile Cells skeletal muscle cells red(slow) white (fast) intermediate muscle spindleXXnuclear bag musclespindleXXnuclear chain satellite cell (stem cell) heart muscle cellsordinary nodal Purkinje fiber smooth muscle cells myoepithelial cells ofiris of exocrine glands Cells of Blood and Immune System red blood cellmegakaryocyte macrophages monocyte connective tissue macrophage(various) Langerhans cell (in epidermis) osteoclast (in bone) dendriticcell (in lymphoid tissues) microglial cell (in central nervous system)neutrophil eosinophil basophil mast cell T lymphocyte helper T cellsuppressor T cell killer T cell B lymphocyte IgM IgG IgA IgE killer cellstem cells for the blood and immune system (various) Sensory Transducersphotoreceptors rod cones blue sensitive green sensitive red sensitivehearing inner hair cell of organ of Corti outer hair cell of organ ofCorti acceleration and gravity type I hair cell of vestibular apparatusof ear type II hair cell of vestibular apparatus of ear taste type 11taste bud cell smell olfactory neuron basal cell of olfactory epithelium(stem cell for olfactory neurons) blood Ph carotid body cell type I typeII touch Merkel cell of epidermis primary sensory neurons specializedfor touch temperature primary sensory neurons specialized fortemperature cold sensitive heat sensitive pain primary sensory neuronsspecialized for pain configurations and forces in musculoskeletal systemproprioceptive primary sensory neurons Autonomic Neurons cholinergicadrenergic peptidergic Supporting Cells of Sense Organs and ofPeripheral Neurons supporting cells of organ of Corti inner pillar cellouter pillar cell inner phalangeal cell outer phalangeal cell bordercell Hensen cell supporting cell of vestibular apparatus supporting cellof taste bud (type I taste bud cell) supporting cell of olfactoryepithelium Schwann cell satellite cell (encapsulating peripheral nervecell bodies) enteric glial cell Neurons and Glial Cells of CentralNervous System neurons glial cells astrocyte oligodendrocyte Lens Cellsanterior lens epithelial cell lens fiber (crystallin-containing cell)Pigment Cells melanocyte, retinal pigmented epithelial cell Germ Cellsoogonium/oocyte spermatocyte spermatogonium (stem cell for spermatocyte)Nurse Cells ovarian follicle cell Sertoli cell (in testis) thymusepithelial cell Stem Cells embryonic stem cell embryonic germ cell adultstem cell fetal stem cell

IX. Diagnostic Assays

A) Introduction

1) TRT Assays

The present invention provides a wide variety of assays for TRT,preferably hTRT, and telomerase. These assays provide, inter alia, thebasis for sensitive, inexpensive, convenient, and widely applicableassays for diagnosis and prognosis of a number of human diseases, ofwhich cancer is an illustrative example. As noted supra, hTRT geneproducts (protein and mRNA) are usually elevated in immortal human cellsrelative to most normal mortal cells (i.e., telomerase-negative cellsand most telomerase-positive normal adult somatic cells). Thus, in oneaspect, the invention provides assays useful for detecting or measuringthe presence, absence, or quantity of an hTRT gene product in a samplefrom, or containing, human or other mammalian or eukayotic cells tocharacterize the cells as immortal (such as a malignant tumor cell) ormortal (such as most normal somatic cells in adults) or as telomerasepositive or negative.

Any condition characterized by the presence or absence of an hTRT geneproduct (i.e., protein or RNA) may be diagnosed using the methods andmaterials described herein. These include, as described more fullybelow, cancers, other diseases of accelerated cell proliferation,immunological disorders, fertility, infertility, and others. Moreover,because the degree to which telomerase activity is elevated in cancercells is correlated with characteristics of the tumor, such asmetastatic potential, monitoring hTRT, mRNA or protein levels can beused to estimate and predict the likely future progression of a tumor.

In one aspect, the diagnostic and prognostic methods of the inventionentail determining whether a human TRT gene product is present in abiological sample (e.g., from a patient). In a second aspect, theabundance of hTRT gene product in a biological sample (e.g., from apatient) is determined and compared to the abundance in a control sample(e.g., normal cells or tissues). In a third aspect, the cellular orintracellular localization of an hTRT gene product is determined in acell or tissue sample. In a fourth aspect, host (e.g., patient) cellsare assayed to identify nucleic acids with sequences characteristic of aheritable propensity for abnormal hTRT gene expression (abnormalquantity, regulation, or product), such as is useful in geneticscreening or genetic counseling. In a fifth aspect, the assays of theinvention are used detect the presence of anti-hTRT antibodies (e.g., inpatient serum). The methods described below in some detail areindicative of useful assays that can be carried out using the sequencesand relationships disclosed herein. However, numerous variations orother applications of these assays will be apparent to those of ordinaryskill in the art in view of this disclosure.

It will be recognized that, although the assays below are presented interms of diagnostic and prognostic methods, they may be used whenever anhTRT gene, gene product, or variant is to be detected, quantified, orcharacterized. Thus, for example, the “diagnostic” methods describedinfra are useful for assays of hTRT or telomerase during production andpurification of hTRT or human telomerase, for characterization of celllines derived from human cells (e.g., to identify immortal lines), forcharacterization of cells, non-human animals, plants, fungi, bacteria orother organisms that comprise a human TRT gene or gene product (orfragments thereof).

As used herein, the term “diagnostic” has its usual meaning ofidentifying the presence or nature of a disease (e.g., cancer),condition (e.g., infertile, activated), or status (e.g., fertile), andthe term “prognostic” has its usual meaning of predicting the probabledevelopment and/or outcome of a disease or condition. Although these twoterms are used in somewhat different ways in a clinical setting, it willbe understood that any of the assays or assay formats disclosed below inreference to “diagnosis” are equally suitable for determination ofprognosis because it is well established that higher telomerase activitylevels are associated with poorer prognoses for cancer patients, andbecause the present invention provides detection methods specific forhTRT, which is expressed at levels that closely correlate withtelomerase activity in a cell.

2) Diagnosis and Prognosis of Cancer

The determination of an hTRT gene, mRNA or protein level above normal orstandard range is indicative of the presence of telomerase-positivecells, or immortal, of which certain tumor cells are examples. Becausecertain embryonic and fetal cells, as well as certain adult stem cells,express telomerase, the present invention also provides methods fordetermining other conditions, such as pregnancy, by the detection orisolation of telomerase positive fetal cells from maternal blood. Thesevalues can be used to make, or aid in making, a diagnosis, even when thecells would not have been classified as cancerous or otherwise detectedor classified using traditional methods. Thus, the methods of thepresent invention permit detection or verification of cancerous or otherconditions associated with telomerase with increased confidence, and atleast in some instances at an earlier stage. The assays of the inventionallow discrimination between different classes and grades of humantumors or other cell-proliferative diseases by providing quantitativeassays for the hTRT gene and gene products and thereby facilitate theselection of appropriate treatment regimens and accurate diagnoses.Moreover, because levels of telomerase activity can be used todistinguish between benign and malignant tumors (e.g., U.S. Pat. No.5,489,508; Hiyama et al., 1997, Proc. Am Ass. Cancer Res. 38:637), topredict immanence of invasion (e.g., U.S. Pat. No. 5,639,613; Yashima etal., 1997, Proc. Am Ass. Cancer Res. 38:326), and to correlate withmetastatic potential (e.g., U.S. Pat. No. 5,648,215; Pandita et al,1996, Proc. Am Ass. Cancer Res. 37:559), these assays will be useful forprophylaxis, detection, and treatment of a wide variety of humancancers.

For prognosis of cancers (or other diseases or conditions characterizedby elevated telomerase), a prognostic value of hTRT gene product (mRNAor protein) or activity for a particular tumor type, class or grade, isdetermined as described infra. hTRT protein or mRNA levels or telomeraseactivity in a patient can also be determined (e.g., using the assaysdisclosed herein) and compared to the prognostic level.

Depending on the assay used, in some cases the abundance of an hTRT geneproduct in a sample will be considered elevated whenever it isdetectable by the assay. Due to the low abundance of hTRT mRNA andprotein even in telomerase-positive cells, and the rarity ornon-existence of these gene products in normal or telomerase-negativecells, sensitive assays are required to detect the hTRT gene product ifpresent at all in normal cells. If less sensitive assays are selected,hTRT gene products will be undetectable in healthy tissue but will bedetectable in telomerase-positive cancer or other telomerase-positivecells. Typically, the amount of hTRT gene product in an elevated sampleis at least about five, frequently at least about ten, more often atleast about 50, and very often at least about 100 to 1000 times higherthan the levels in telomerase-negative control cells or cells fromhealthy tissues in an adult, where the percentage of telomerase-positivenormal cells is very low.

The diagnostic and prognostic methods of the present invention can beemployed with any cell or tissue type of any origin and can be used todetect an immortal or neoplastic cell, or tumor tissue, or cancer, ofany origin. Types of cancer that may be detected include, but are notlimited to, all those listed supra in the discussion of therapeuticapplications of hTRT.

The assays of the invention are also useful for monitoring the efficacyof therapeutic intervention in patients being treated with anticancerregimens. Anticancer regimens that can be monitored include allpresently approved treatments (including chemotherapy, radiationtherapy, and surgery) and also includes treatments to be approved in thefuture, such as telomerase inhibition or activation therapies asdescribed herein. (See, e.g., See PCT Publication Nos. 96/01835 and96/40868 and U.S. Pat. No. 5,583,016; all of which are incorporated byreference in their entirety).

In another aspect, the assays described below are useful for detectingcertain variations in hTRT gene sequence (mutations and heritable hTRTalleles) that are indicative of a predilection for cancers or otherconditions associated with abnormal regulation of telomerase activity(infertility, premature aging).

3) Diagnosis of Conditions Other than Cancer

In addition to diagnosis of cancers, the assays of the present inventionhave numerous other applications. The present invention providesreagents and methods/diagnosis of conditions or diseases characterizedby under- or over-expression of telomerase or hTRT gene products incells. In adults, a low level of telomerase activity is normally foundin a limited complement of normal human somatic cells, e.g., stem cells,activated lymphocytes and germ cells, and is absent from other somaticcells. Thus, the detection of hTRT or telomerase activity in cells inwhich it is normally absent or inactive, or detection at abnormal (i.e.,higher or lower than normal) levels in cells in which hTRT is normallypresent at a low level (such as stem cells, activated lymphocytes andgerm cells), can be diagnostic of a telomerase-related disease orcondition or used to identify or isolate a specific cell type (i.e., toisolate stem cells). Examples of such diseases and conditions include:diseases of cell proliferation, immunological disorders, infertility,diseases of immune cell function, pregnancy, fetal abnormalities,premature aging, and others. Moreover, the assays of the invention areuseful for monitoring the effectiveness of therapeutic intervention(including but not limited to drugs that modulate telomerase activity)in a patient or in a cell- or animal-based assay.

In one aspect, the invention provides assays useful for diagnosinginfertility. Human germ cells (e.g., spermatogonia cells, theirprogenitors or descendants) are capable of indefinite proliferation andcharacterized by high telomerase activity. Abnormal levels or productsor diminished levels of hTRT gene products can result in inadequate orabnormal production of spermatozoa, leading to infertility or disordersof reproduction. Accordingly, the invention provides assays (methods andreagents) for diagnosis and treatment of “telomerase-based” reproductivedisorders. Similarly, the assays can be used to monitor the efficacy ofcontraceptives (e.g., male contraceptives) that target or indirectlyaffect sperm production (and which would reduce hTRT levels ortelomerase activity).

In another aspect, the invention provides assays for analysis oftelomerase and hTRT levels and function in stem cells, fetal cells,embryonic cells, activated lymphocytes and hematopoietic stem cells. Forexample, assays for hTRT gene product detection can be used to monitorimmune function generally (e.g., by monitoring the prevalence ofactivated lymphocytes or abundance of progenitor stem cells), toidentify or select or isolate activated lymphocytes or stem cells (basedon elevated hTRT levels), and to monitor the efficacy of therapeuticinterventions targeting these tissues (e.g., immunosuppressive agents ortherapeutic attempt to expand a stem cell population).

The invention also provides assays useful for identification ofanti-telomerase and anti-TRT immunoglobulins (found in serum from apatient). The materials and assays described herein can be used toidentify patients in which such autoimmune antibodies are found,permitting diagnosis and treatment of the condition associated with theimmunoglobulins.

4) Monitoring Cells in Culture

The assays described herein are also useful for monitoring theexpression of hTRT gene products and characterization of hTRT genes incells ex vivo or in vitro. Because elevated hTRT levels arecharacteristic of immortalized cells, the assays of the invention can beused, for example, to screen for, or identify, immortalized cells or toidentify an agent capable of mortalizing immortalized cells byinhibiting hTRT expression or function. For example, the assay will beuseful for identifying cells immortalized by increased expression ofhTRT in the cell, e.g., by the expression of a recombinant hTRT or byincreased expression of an endogenously coded hTRT (e.g., by promoteractivation).

Similarly, these assays may be used to monitor hTRT expression intransgenic animals or cells (e.g., yeast or human cells containing anhTRT gene). In particular, the effects of certain treatments (e.g.,application of known or putative telomerase antagonists) on the hTRTlevels in human and nonhuman cells expressing the hTRT of the inventioncan be used for identifying useful drugs and drug candidates (e.g.,telomerase activity-modulating drugs).

B) Normal, Diagnostic, and Prognostic Values

Assays for the presence or quantity of hTRT gene products may be carriedout and the results interpreted in a variety of ways, depending on theassay format, the nature of the sample being assayed, and theinformation sought. For example, the steady state abundance of hTRT geneproducts is so low in most human somatic tissues that they areundetectable by certain assays. Moreover, there is generally notelomerase activity in the cells of these tissues, making verificationof activity quite easy. Conversely, hTRT protein and/or hTRT mRNA ortelomerase is sufficiently abundant in other telomerase-positivetissues, e.g., malignant tumors, so that the same can be detected usingthe same assays. Even in those somatic cell types in which low levels oftelomerase activity can normally be detected (e.g., stem cells, andcertain activated hematopoietic system cells), the levels of hTRT mRNAand telomerase activity are a small fraction (e.g., estimated at about1% or less) of the levels in immortal cells; thus, immortal and mortalcells may be easily distinguished by the methods of the presentinvention. It will be appreciated that, when a “less sensitive” assay isused, the mere detection of the hTRT gene product in a biological samplecan itself be diagnostic, without the requirement for additionalanalysis. Moreover, although the assays described below can be madeexquisitely sensitive, they may also, if desired, be made less sensitive(e.g., through judicious choice of buffers, wash conditions, numbers ofrounds of amplification, reagents, and/or choice of signal amplifiers).Thus, virtually any assay can be designed so that it detects hTRT geneproducts only in biological samples in which they are present at aparticular concentration, e.g. a higher concentration than in healthy orother control tissue. In this case, any detectable level of hTRT mRNA orprotein will be considered elevated in cells from post-natal humansomatic tissue (other than hematopoietic cells and other stem cells).

In some cases, however, it will be desirable to establish normal orbaseline values (or ranges) for hTRT gene product expression levels,particularly when very sensitive assays capable of detecting very lowlevels of hTRT gene products that may be present in normal somatic cellsare used. Normal levels of expression or normal expression products canbe determined for any particular population, subpopulation, or group oforganisms according to standard methods well known to those of skill inthe art and employing the methods and reagents of the invention.Generally, baseline (normal) levels of hTRT protein or hTRT mRNA aredetermined by quantitating the amount of hTRT protein and/or mRNA inbiological samples (e.g., fluids, cells or tissues) obtained from normal(healthy) subjects, e.g., a human subject. For certain samples andpurposes, one may desire to quantitate the amount of hTRT gene producton a per cell, or per tumor cell, basis. To determine the cellularity ofa sample, one may measure the level of a constitutively expressed geneproduct or other gene product expressed at known levels in cells of thetype from which the sample was taken. Alternatively, normal values ofhTRT protein or hTRT mRNA can be determined by quantitating the amountof hTRT protein/RNA in cells or tissues known to be healthy, which areobtained from the same patient from whom diseased (or possibly diseased)cells are collected or from a healthy individual. Alternatively,baseline levels can be defined in some cases as the level present innon-immortal human somatic cells in culture. It is possible that normal(baseline) values may differ somewhat between different cell types (forexample, hTRT mRNA levels will be higher in testis than kidney), oraccording to the age, sex, or physical condition of a patient. Thus, forexample, when an assay is used to determine changes in hTRT levelsassociated with cancer, the cells used to determine the normal range ofhTRT gene product expression can be cells from persons of the same or adifferent age, depending on the nature of the inquiry. Application ofstandard statistical methods used in molecular genetics permitsdetermination of baseline levels of expression, as well as permitsidentification of significant deviations from such baseline levels.

In carrying out the diagnostic and prognostic methods of the invention,as described above, it will sometimes be useful to refer to “diagnostic”and “prognostic values” As used herein, “diagnostic value” refers to avalue that is determined for the hTRT gene product detected in a samplewhich, when compared to a normal (or “baseline”) range of the hTRT geneproduct is indicative of the presence of a disease. The disease may becharacterized by high telomerase activity (e.g., cancer), the absence oftelomerase activity (e.g., infertility), or some intermediate value.“Prognostic value” refers to an amount of the hTRT gene product detectedin a given cell type (e.g., malignant tumor cell) that is consistentwith a particular diagnosis and prognosis for the disease (e.g.,cancer). The amount (including a zero amount) of the hTRT gene productdetected in a sample is compared to the prognostic value for the cellsuch that the relative comparison of the values indicates the presenceof disease or the likely outcome of the disease (e.g., cancer)progression. In one embodiment, for example, to assess tumor prognosis,data are collected to obtain a statistically significant correlation ofhTRT levels with different tumor classes or grades. A predeterminedrange of hTRT levels is established for the same cell or tissue sampleobtained from subjects having known clinical outcomes. A sufficientnumber of measurements is made to produce a statistically significantvalue (or range of values) to which a comparison will be made. Thepredetermined range of hTRT levels or activity for a given cell ortissue sample can then be used to determine a value or range for thelevel of hTRT gene product that would correlate to favorable (or lessunfavorable) prognosis (e.g., a “low level” in the case of cancer). Arange corresponding to a “high level” correlated to an (or a more)unfavorable prognosis in the case of cancer can similarly be determined.The level of hTRT gene product from a biological sample (e.g., a patientsample) can then be determined and compared to the low and high rangesand used to predict a clinical outcome.

Although the discussion above refers to cancer for illustration, it willbe understood that diagnostic and prognostic values can also bedetermined for other diseases (e.g., diseases of cell proliferation) andconditions and that, for diseases or conditions other than cancer, a“high” level may be correlated with the desired outcome and a “low”level correlated with an unfavorable outcome. For example, some diseasesmay be characterized by a deficiency (e.g., low level) of telomeraseactivity in stem cells, activated lymphocytes, or germline cells. Insuch cases, “high” levels of hTRT gene products relative to cells ofsimilar age and/or type (e.g., from other patients or other tissues in aparticular patient) may be correlated with a favorable outcome.

It will be appreciated that the assay methods do not necessarily requiremeasurement of absolute values of hTRT, unless it is so desired, becauserelative values are sufficient for many applications of the methods ofthe present invention. Where quantitation is desirable, the presentinvention provides reagents such that virtually any known method forquantitating gene products can be used.

The assays of the invention may also be used to evaluate the efficacy ofa particular therapeutic treatment regime in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient. Inthese cases, it may be desirable to establish the baseline for thepatient prior to commencing therapy and to repeat the assays one or moretimes through the course of treatment, usually on a regular basis, toevaluate whether hTRT levels are moving toward the desired endpoint(e.g., reduced expression of hTRT when the assay is for cancer) as aresult of the treatment.

One of skill will appreciate that, in addition to the quantity orabundance of hTRT gene products, variant or abnormal expression patterns(e.g., abnormal amounts of RNA splicing variants) or variant or abnormalexpression products (e.g., mutated transcripts, truncated or non-sensepolypeptides) may also be identified by comparison to normal expressionlevels and normal expression products. In these cases determinationof“normal” or “baseline” involves identifying healthy organisms and/ortissues (i.e. organisms and/or tissues without hTRT expressiondisregulation or neoplastic growth) and measuring expression levels ofthe variant hTRT gene products (e.g., splicing variants), or sequencingor detecting the hTRT gene, mRNA, or reverse transcribed cDNA to obtainor detect typical (normal) sequence variations. Application of standardstatistical methods used in molecular genetics permits determination ofsignificant deviations from such baseline levels.

C) Detection and Quantitation of TRT Gene Products

As has been emphasized herein, hTRT gene products are usually found inmost normal somatic cells at extremely low levels. For example, the mRNAencoding hTRT protein is extremely rare or absent in alltelomerase-negative cell types studied thus far. In immortal cells, suchas 293 cells, HTRT mRNA may be present at only about 100 copies percell, while normal somatic cells may have as few as one or zero copiesper cell. It will thus be apparent that, when highly sensitive assaysfor hTRT gene products are desired, it will sometimes be advantageous toincorporate signal or target amplification technologies into the assayformat. See, for example, Plenat et al., 1997, Ann. Pathol. 17:17(fluoresceinyl-tyramide signal amplification); Zehbe et al., 1997, J.Pathol. 150:1553 (catalyzed reporter deposition); other referenceslisted herein (e.g., for bDNA signal amplification, for PCR and othertarget amplification formats); and other techniques known in the art.

As noted above, it is often unnecessary to quantitate the hTRT mRNA orprotein in the assays disclosed herein, because the detection of an hTRTgene product (under assay conditions in which the product is notdetectable in control, e.g., telomerase-negative cells) is in itselfsufficient for a diagnosis. As another example, when the levels ofproduct found in a test (e.g., tumor) and control (e.g., healthy cell)samples are directly compared, quantitation may be superfluous.

When desired, however, quantities of hTRT gene product measured in theassays described herein may be described in a variety of ways, dependingon the method of measurement and convenience. Thus, normal, diagnostic,prognostic, high or low quantities of hTRT protein/mRNA may be expressedas standard units of weight per quantity of biological sample (e.g.,picograms per gram tissue, picograms per 10¹² cells), as a number ofmolecules per quantity of biological sample (e.g., transcripts/cell,moles/cell), as units of activity per cell or per other unit quantity,or by similar methods. The quantity of hTRT gene product can also beexpressed in relation to the quantity of another molecule; examplesinclude: number of hTRT transcripts in sample/number of 28S rRNAtranscripts in sample; nanograms of hTRT protein/ nanograms of totalprotein; and the like.

When measuring hTRT gene products in two (or more) different samples, itwill sometimes be useful to have a common basis of comparison for thetwo samples. For example, when comparing a sample of normal tissue and asample of cancerous tissue, equal amounts of tissue (by weight, volume,number of cells, etc.) can be compared. Alternatively, equivalents of amarker molecule (e.g., 28S rRNA, hTR, telomerase activity, telomerelength, actin) may be used. For example, the amount of hTRT protein in ahealthy tissue sample containing 10 picograms of 28S rRNA can becompared to a sample of diseased tissue containing the same amount of28S rRNA.

It will also be recognized by those of skill that virtually any of theassays described herein can be designed to be quantitative. Typically, aknown quantity or source of an hTRT gene product (e.g., produced usingthe methods and compositions of the invention) is used to calibrate theassay.

In certain embodiments, assay formats are chosen that detect thepresence, absence, or abundance of an hTRT allele or gene product ineach cell in a sample (or in a representative sampling). Examples ofsuch formats include those that detect a signal by histology (e.g.,immunohistochemistry with signal-enhancing or target-enhancingamplification steps) or fluorescence-activated cell analysis or cellsorting (FACS). These formats are particularly advantageous when dealingwith a highly heterogeneous cell population (e.g., containing multiplecells types in which only one or a few types have elevated hTRT levels,or a population of similar cells expressing telomerase at differentlevels).

D) Sample Collection

The hTRT gene or gene product (i.e., mRNA or polypeptide) is preferablydetected and/or quantified in a biological sample. Such samples include,but are not limited to, cells (including whole cells, cell fractions,cell extracts, and cultured cells or cell lines), tissues (includingblood, blood cells (e.g., white cells), and tissue samples such as fineneedle biopsy samples (e.g., from prostate, breast, thyroid, etc.)),body fluids (e.g., urine, sputum, amniotic fluid, blood, peritonealfluid, pleural fluid, semen) or cells collected therefrom (e.g., bladdercells from urine, lymphocytes from blood), media (from cultured cells orcell lines), and washes (e.g., of bladder and lung). Biological samplesmay also include sections of tissues such as frozen sections taken forhistological purposes. For cancer diagnosis and prognosis, a sample willbe obtained from a cancerous or precancerous or suspected canceroustissue or tumor. It will sometimes be desirable to freeze a biologicalsample for later analysis (e.g., when monitoring efficacy of drugtreatments).

In some cases, the cells or tissues may be fractionated before analysis.For example, in a tissue biopsy from a patient, a cell sorter (e.g., afluorescence-activated cell sorter) may be used to sort cells accordingto characteristics such as expression of a surface antigen (e.g., atumor specific antigen) according to well known methods.

Although the sample is typically taken from a human patient or cellline, the assays can be used to detect hTRT homolog genes or geneproducts in samples from other animals. Alternatively, hTRT genes andgene products can be assayed in transgenic animals or organismsexpressing a human TRT protein or nucleic acid sequence.

The sample may be pretreated as necessary by dilution in an appropriatebuffer solution or concentrated, if desired. Any of a number of standardaqueous buffer solutions, employing one of a variety of buffers, such asphosphate, Tris-buffer, or the like, at physiological pH can be used.

A “biological sample” obtained from a patient can be referred to eitheras a “biological sample” or a “patient sample.” It will be appreciatedthat analysis of a “patient sample” need not necessarily require removalof cells or tissue from the patient. For example, appropriately labeledhTRT-binding agents (e.g., antibodies or nucleic acids) can be injectedinto a patient and visualized (when bound to the target) using standardimaging technology (e.g., CAT, NMR, and the like.)

E) Nucleic Acid Assays

In one embodiment, this invention provides for methods of detectingand/or quantifying expression of hTRT mRNAs (including splicing orsequence variants and alternative alleles). In an alternativeembodiment, the invention provides methods for detecting and analyzingnormal or abnormal hTRT genes (or fragments thereof). The form of suchqualitative or quantitative assays may include, but is not limited to,amplification-based assays with or without signal amplification,hybridization based assays, and combination amplification-hybridizationassays. It will be appreciated by those of skill that the distinctionbetween hybridization and amplification is for convenience only: asillustrated in the examples below, many assay formats involve elementsof both hybridization and amplification, so that the categorization issomewhat arbitrary in some cases.

1) Preparation of Nucleic Acids

In some embodiments, nucleic acid assays are performed with a sample ofnucleic acid isolated from the cell, tissue, organism, or cell line tobe tested. The nucleic acid (e.g., genomic DNA, RNA or cDNA) may be“isolated” from the sample according to any of a number of methods wellknown to those of skill in the art. In this context, “isolated” refersto any separation of the species or target to be detected from any othersubstance in the mixture, but does not necessarily indicate asignificant degree of purification of the target. One of skill willappreciate that, where alterations in the copy number of the hTRT geneare to be detected, genomic DNA is the target to be detected.Conversely, where expression levels of a gene or genes are to bedetected, RNA is the target to be detected in a nucleic acid-basedassay. In one preferred embodiment, the nucleic acid sample is the totalmRNA (i.e., poly(A)⁺ RNA) in a biological sample. Methods for isolatingnucleic acids are well known to those of skill in the art and aredescribed, for example, Tijssen, P. ed. of Laboratory Techniques inBiochemistry and Molecular Biology: Hybridization with Nucleic AcidProbes, Part I. Theory and Nucleic Acid Preparation, Elsevier, N.Y.(1993) Chapt. 3, which is incorporated herein by reference. In oneembodiment, the total nucleic acid is isolated from a given sample usingan acid guanidinium-phenol-chloroform extraction method and poly(A)+mRNA is isolated by oligo-dT column chromatography or by using (dT)nmagnetic beads (see, e.g., Sambrook et al., and Ausubel et al., supra).

In alternative embodiments, it is not necessary to isolate nucleic acids(e.g., total or polyA⁺ RNA) from the biological sample prior to carryingout amplification, hybridization or other assays. These embodiments havecertain advantages when hTRT RNA is to be measured, because they reducethe possibility of loss of hTRT mRNA during isolation and handling. Forexample, many amplification techniques such as PCR and RT-PCR definedabove can be carried out using permeabilized cells (histologicalspecimens and FACS analyses), whole lysed cells, or crude cell fractionssuch as certain cell extracts. Preferably, steps are taken to preservethe integrity of the target nucleic acid (e.g., mRNA) if necessary(e.g., addition of RNAase inhibitors). Amplification and hybridizationassays can also be carried out in situ, for example, in thin tissuesections from a biopsy sample or from a cell monolayer (e.g., bloodcells or disagregated tissue culture cells). Amplification can also becarried out in an intact whole cell or fixed cells. For example, PCR,RT-PCR, or LCR amplification methods may be carrier out, as is wellknown in the art, in situ, e.g., using a polymerase or ligase, a primeror primer(s), and (deoxy)ribonucleoside triphosphates (if a polymeraseis employed), and reverse transcriptase and primer (if RNA is to betranscribed and the cDNA is to be detected) on fixed, permeabilized, ormicroinjected cells to amplify target hTRT RNA or DNA. Cells containinghTRT RNA (e.g., telomerase positive cells) or an hTRT DNA sequence ofinterest can then be detected. This method is often useful whenfluorescently-labeled dNTPs, primers, or other components are used inconjunction with microscopy, FACS analysis or the equivalent.

2) Amplification Based Assays

In one embodiment, the assays of the present invention areamplification-based assays for detection of an hTRT gene or geneproduct. In an amplification based assay, all or part of an hTRT gene ortranscript (e.g., mRNA or cDNA; hereinafter also referred to as“target”) is amplified, and the amplification product is then detecteddirectly or indirectly. When there is no underlying gene or gene productto act as a template, no amplification product is produced (e.g., of theexpected size), or amplification is non-specific and typically there isno single amplification product. In contrast, when the underlying geneor gene product is present, the target sequence is amplified, providingan indication of the presence and/or quantity of the underlying gene ormRNA. Target amplification-based assays are well known to those of skillin the art.

The present invention provides a wide variety of primers and probes fordetecting hTRT genes and gene products. Such primers and probes aresufficiently complementary to the hTRT gene or gene product to hybridizeto the target nucleic acid. Primers are typically at least 6 bases inlength, usually between about 10 and about 100 bases, typically betweenabout 12 and about 50 bases, and often between about 14 and about 25bases in length. One of skill, having reviewed the present disclosure,will be able, using routine methods, to select primers to amplify all,or any portion, of the hTRT gene or gene product, or to distinguishbetween variant gene products, hTRT alleles, and the like. Table 2 listsillustrative primers useful for PCR amplification of the hTRT, orspecific hTRT gene products or regions. As is known in the art, singleoligomers (e.g., U.S. Pat. No. 5,545,522), nested sets of oligomers, oreven a degenerate pool of oligomers may be employed for amplification,e.g., as illustrated by the amplification of the Tetrahymena TRT cDNA asdescribed infra.

The invention provides a variety of methods for amplifying and detectingan hTRT gene or gene product, including the polymerase chain reaction(including all variants, e.g., reverse-transcriptase-PCR; the SunriseAmplification System (Oncor, Inc, Gaithersburg Md.); and numerous othersknown in the art). In one illustrative embodiment, PCR amplification iscarried out in a 50 μl solution containing the nucleic acid sample(e.g., cDNA obtained through reverse transcription of hTRT RNA), 100 μMin each dNTP (dATP, dCTP, dGTP and dTTP; Pharmacia LKB Biotechnology,N.J.), the hTRT-specific PCR primer(s), 1 unit/Taq polymerase (PerkinElmer, Norwalk Conn.), 1×PCR buffer (50 mM KCl, 10 mM Tris, pH 8.3 atroom temperature, 1.5 mM MgCl₂, 0.01% gelatin) with the amplificationran for about 30 cycles at 94° for 45 sec, 55° for 45 sec and 72° for 90sec. However, as will be appreciated, numerous variations may be made tooptimize the PCR amplification for any particular reaction.

Other suitable target amplification methods include the ligase chainreaction (LCR; e.g., Wu and Wallace, 1989, Genomics 4:560; Landegren etal., 1988, Science, 241: 1077, Barany, 1991, Proc. Natl. Acad. Sci. USA88:189 and Barringer et al., 1990, Gene, 89: 117); strand displacementamplification (SDA; e.g., Walker et al., 1992, Proc. Natl. Acad. Sci.U.S.A. 89:392-396); transcription amplification (e.g., Kwoh et al.,1989, Proc. Natl. Acad. Sci. USA, 86: 1173); self-sustained sequencereplication (3SR; e.g., Fahy et al., 1992, PCR Methods Appl. 1:25,Guatelli et al., 1990, Proc. Nat. Acad. Sci. USA, 87: 1874); the nucleicacid sequence based amplification (NASBA, Cangene, Mississauga, Ontario;e.g., Compton, 1991, Nature 350:91); the transcription-basedamplification system (TAS); and the self-sustained sequence replicationsystem (SSR). Each of the aforementioned publications is incorporatedherein by reference. One useful variant of PCR is PCR ELISA (e.g.,Boehringer Mannheim Cat. No. 1 636 111) in which digoxigenin-dUTP isincorporated into the PCR product. The PCR reaction mixture is denaturedand hybridized with a biotin-labeled oligonucleotide designed to annealto an internal sequence of the PCR product. The hybridization productsare immobilized on streptavidin coated plates and detected usinganti-digoxigenin antibodies. Examples of techniques sufficient to directpersons of skill through in vitro amplification methods are found in PCRTechnology: Principles and Applications for DNA Amplification, H.Erlich, Ed. Freeman Press, New York, N.Y. (1992); PCR Protocols: A Guideto Methods and Applications, eds. Innis, Gelfland, Snisky, and White,Academic Press, San Diego, Calif. (1990); Mattila et al., 1991, NucleicAcids Res. 19: 4967; Eckert and Kunkel, (1991) PCR Methods andApplications 1: 17; PCR, eds. McPherson, Quirkes, and Taylor, IRL Press,Oxford; U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188; Barringer etal., 1990, Gene, 89:117; Lomell et al., 1989, J. Clin. Chem., 35:1826,each of which is incorporated herein for all purposes.

Amplified products may be directly analyzed, e.g., by size as determinedby gel electrophoresis; by hybridization to a target nucleic acidimmobilized on a solid support such as a bead, membrane, slide, or chip;by sequencing; immunologically, e.g., by PCR-ELISA, by detection of afluorescent, phosphorescent, or radioactive signal; or by any of avariety of other well-known means. For example, an illustrative exampleof a detection method uses PCR primers augmented with hairpin loopslinked to fluorescein and a benzoic acid derivative that serves as aquencher, such that fluorescence is emitted only when the primers unfoldto bind their targets and replication occurs.

Because hTRT mRNA is typically expressed as an extremely raretranscript, present at very low levels even in telomerase positivecells, it is often desirable to optimize or increase the signalresulting from the amplification step. One way to do this is to increasethe number of cycles of amplification. For example, although 20-25cycles are adequate for amplification of most mRNAs using the polymerasechain reaction under standard reaction conditions, detection of hTRTmRNA in many samples can require as many as 30 to 35 cycles ofamplification, depending on detection format and efficiency ofamplification. It will be recognized that judicious choice of theamplification conditions including the number of amplification cyclescan be used to design an assay that results in an amplification productonly when there is a threshold amount of target in the test sample(i.e., so that only samples with a high level of hTRT mRNA give a“positive” result). In addition, methods are known to increase signalproduced by amplification of the target sequence. Methods for augmentingthe ability to detect the amplified target include signal amplificationsystem such as: branched DNA signal amplification (e.g., U.S. Pat. No.5,124,246; Urdea, 1994, Bio/Tech. 12:926); tyramide signal amplification(TSA) system (Du Pont); catalytic signal amplification (CSA; Dako); QBeta Replicase systems (Tyagi et al., 1996, Proc. Nat. Acad. Sci. USA,93: 5395); or the like.

One of skill in the art will appreciate that whatever amplificationmethod is used, a variety of quantitative methods known in the art canbe used if quantitation is desired. For example, when desired, two ormore polynucleotides can be co-amplified in a single sample. This methodcan be used as a convenient method of quantitating the amount of hTRTmRNA in a sample, because the reverse transcription and amplificationreactions are carried out in the same reaction for a target and controlpolynucleotide. The co-amplification of the control polynucleotide(usually present at a known concentration or copy number) can be usedfor normalization to the cell number in the sample as compared to theamount of hTRT in the sample. Suitable control polynucleotides forco-amplification reactions include DNA, RNA expressed from housekeepinggenes, constitutively expressed genes, and in vitro synthesized RNAs orDNAs added to the reaction mixture. Endogenous control polynucleotidesare those that are already present in the sample, while exogenouscontrol polynucleotides are added to a sample, creating a “spiked”reaction. Illustrative control RNAs include β-actin RNA, GAPDH RNA,snRNAs, hTR, and endogenously expressed 28S rRNA (see Khan et al., 1992,Neurosci. Lett. 147:114). Exogenous control polynucleotides include asynthetic AW106 cRNA, which may be synthesized as a sense strand frompAW106 by T7 polymerase. It will be appreciated that for theco-amplification method to be useful for quantitation, the control andtarget polynucleotides must typically both be amplified in a linearrange. Detailed protocols for quantitative PCR may be found in PCRProtocols, A Guide to Methods and Applications, Innis et al., AcademicPress, Inc. N.Y., (1990) and Ausubel et al., supra (Unit 15) and Diaco,R. (1995) Practical Considerations for the Design of Quantitative PCRAssays, in PCR Strategies, pg. 84-108, Innis et al. eds, Academic Press,New York.

Depending on the sequence of the endogenous or exogenous standard,different primer sets may be used for the co-amplification reaction. Inone method, called competitive amplification, quantitative PCR involvessimultaneously co-amplifying a known quantity of a control sequenceusing the same primers used for amplification of the target nucleic acid(one pair of 2 primers). In an alternative embodiment, known asnon-competitive competition, the control sequence and the targetsequence (e.g., hTRT cDNA) are amplified using different primers (i.e.,2 pairs of 2 primers). In another alternative embodiment, calledsemi-competitive amplification, three primers are used, one of which ishTRT-specific, one of which is control specific, and one of which iscapable of annealing to both the target and control sequences.Semi-competitive amplification is described in U.S. Pat. No. 5,629,154,which is incorporated herein by reference.

3) Hybridization-Based Assays

a) Generally

A variety of methods for specific DNA and RNA measurement using nucleicacid hybridization techniques are known to those of skill in the art(see Sambrook et al., supra). Hybridization based assays refer to assaysin which a probe nucleic acid is hybridized to a target nucleic acid.Usually the nucleic acid hybridization probes of the invention areentirely or substantially identical to a contiguous sequence of the hTRTgene or RNA sequence. Preferably, nucleic acid probes are at least about10 bases, often at least about 20 bases, and sometimes at least about200 bases or more in length. Methods of selecting nucleic acid probesequences for use in nucleic acid hybridization are discussed inSambrook et al., supra. In some formats, at least one of the target andprobe is immobilized. The immobilized nucleic acid may be DNA, RNA, oranother oligo- or poly-nucleotide, and may comprise natural ornon-naturally occurring nucleotides, nucleotide analogs, or backbones.Such assays may be in any of several formats including: Southern,Northern, dot and slot blots, high-density polynucleotide oroligonucleotide arrays (e.g., GeneChips™ Affymetrix), dip sticks, pins,chips, or beads. All of these techniques are well known in the art andare the basis of many commercially available diagnostic kits.Hybridization techniques are generally described in Hames et al., ed.,Nucleic Acid Hybridization, A Practical Approach IRL Press, (1985); Galland Pardue Proc. Natl. Acad. Sci., U.S.A., 63: 378-383 (1969); and Johnet al., Nature, 223: 582-587 (1969).

A variety of nucleic acid hybridization formats are known to thoseskilled in the art. For example, one common format is directhybridization, in which a target nucleic acid is hybridized to alabeled, complementary probe. Typically, labeled nucleic acids are usedfor hybridization, with the label providing the detectable signal. Onemethod for evaluating the presence, absence, or quantity of hTRT mRNA iscarrying out a Northern transfer of RNA from a sample and hybridizationof a labeled hTRT specific nucleic acid probe, as illustrated in Example2. As was noted supra, hTRT mRNA, when present at all, is present invery low quantities in most cells. Therefore, when Northernhybridization is used, it will often be desirable to use anamplification step (or, alternatively, large amounts of starting RNA). Auseful method for evaluating the presence, absence, or quantity of DNAencoding hTRT proteins in a sample involves a Southern transfer of DNAfrom a sample and hybridization of a labeled hTRT specific nucleic acidprobe.

Other common hybridization formats include sandwich assays andcompetition or displacement assays. Sandwich assays are commerciallyuseful hybridization assays for detecting or isolating nucleic acidsequences. Such assays utilize a “capture” nucleic acid covalentlyimmobilized to a solid support and a labeled “signal” nucleic acid insolution. The biological or clinical sample will provide the targetnucleic acid. The “capture” nucleic acid and “signal” nucleic acid probehybridize with the target nucleic acid to form a “sandwich”hybridization complex. To be effective, the signal nucleic acid cannothybridize with the capture nucleic acid.

b) Chip-Based and Slide-Based Assays

The present invention also provides probe-based hybridization assays forhTRT gene products employing arrays of immobilized oligonucleotide orpolynucleotides to which an hTRT nucleic acid can hybridize (i.e., tosome, but usually not all or even most, of the immobilized oligo- orpoly-nucleotides). High density oligonucleotide arrays or polynucleotidearrays provide a means for efficiently detecting the presence andcharacteristics (e.g., sequence) of a target nucleic acid (e.g., hTRTgene, mRNA, or cDNA). Techniques are known for producing arrayscontaining thousands of oligonucleotides complementary to definedsequences, at defined locations on a surface using photolithographictechniques for synthesis in situ (see, e.g., U.S. Pat. Nos. 5,578,832;5,556,752; and 5,510,270; Fodor et al., 1991, Science 251:767; Pease etal., 1994, Proc. Natl. Acad. Sci. USA 91:5022; and Lockhart et al.,1996, Nature Biotech 14:1675) or other methods for rapid synthesis anddeposition of defined oligonucleotides (Blanchard et al., 1996,Biosensors & Bioelectronics 11:687). When these methods are used,oligonucleotides (e.g., 20-mers) of known sequence are synthesizeddirectly on a surface such as a derivatized glass slide. Usually, thearray produced is redundant, having several oligonucleotide probes onthe chip specific for the hTRT polynucleotide to be detected.

Combinations of oligonucleotide probes can be designed to detectalternatively spliced mRNAs, or to identify which of various hTRTalleles is expressed in a particular sample.

In one illustrative embodiment, cDNA prepared by reverse transcriptionof total RNA from a test cell is amplified (e.g., using PCR). Typicallythe amplification product is labeled, e.g., by incorporation of afluorescently labeled dNTP. The labeled cDNAs are then hybridized to achip comprising oligonucleotide probes complementary to varioussubsequences of the hTRT gene. The positions of hybridization aredetermined (e.g., in accordance with the general methods of Shalon etal., 1996, Genome Research 6:639 or Schena et al., 1996, Genome Res.6:639), and sequence (or other information) deduced from thehybridization pattern, by means well known in the art.

In one embodiment, two cDNA samples, each labeled with a differentfluorescent group, are hybridized to the same chip. The ratio of thehybridization of each labeled sample to sites complementary to the hTRTgene are then assayed. If both samples contain the same amount of hTRTmRNA, the ratio of the two fluors will be 1:1 (it will be appreciatedthat the signal from the fluors may need to be adjusted to account forany difference in the molar sensitivity of the fluors). In contrast, ifone sample is from a healthy (or control) tissue and the second sampleis from a cancerous tissue the fluor used in the second sample willpredominate.

c) In Situ Hybridization

An alternative means for detecting expression of a gene encoding an hTRTprotein is in situ hybridization. In situ hybridization assays are wellknown and are generally described in Angerer et al., Methods Enzymol.,152: 649-660 (1987) and Ausubel et al., supra. In an in situhybridization assay, cells or tissue specimens are fixed to a solidsupport, typically in a permeablilized state, typically on a glassslide. The cells are then contacted with a hybridization solution at amoderate temperature to permit annealing of labeled nucleic acid probes(e.g., ³⁵S-labeled riboprobes, fluorescently labeled probes) completelyor substantially complementary to hTRT. Free probe is removed by washingand/or nuclease digestion, and bound probe is visualized directly on theslide by autoradiography or an appropriate imaging techniques, as isknown in the art.

4) Specific Detection of Variants

As noted supra and illustrated in the Examples (e.g., Example 9),amplification primers or probes can be selected to provide amplificationproducts that span specific deletions, truncations, and insertions,thereby facilitating the detection of specific variants or abnormalitiesin the hTRT mRNA.

One example of an hTRT variant gene product that may be detected is anhTRT RNA such as a product (SEQ ID NO:4) described supra and in Example9. The biological function, if any, of the Δ182 variant(s) is not known;however, the truncated hTRT protein putatively encoded by the variantmay be involved in regulation of telomerase activity, e.g., byassembling a non-functional telomerase RNP that titrates telomerasecomponents. Alternatively, negative regulation of telomerase activitycould be accomplished by directing hTRT pre-mRNA (nascent mRNA)processing in a manner leading to elimination of the full length mRNAand reducing hTRT mRNA levels and increasing Δ182 hTRT RNA levels. Forthese and other reasons, the ability to detect Δ182 variants is useful.In addition, it will sometimes be desirable, in samples in which twospecies of hTRT RNA are present (such as a Δ182 hTRT RNA and hTRT RNAencoding the full-length hTRT protein) to compare their relative and/orabsolute abundance.

The invention provides a variety of methods for detection of Δ182variants. For example, amplification using primer pairs spanning the 182basepair deletion will result in different sized products correspondingto the deleted and undeleted hTRT RNAs, if both are present, which canbe distinguished on the basis of size (e.g., by gel electrophoresis).Examples of primer pairs useful for amplifying the region spanning the182 bp deletion include TCP1.14 and TCP1.15 (primer set 1), or TCP1.25and billTCP6 (primer set 2) (see Table 2). These primer pairs can beused individually or in a nested PCR experiment where primer set 1 isused first. It will also be apparent to one of skill that hybridizationmethods (e.g., Northern hybridization) or RNAse protection assays usingan hTRT nucleic acid probe of the invention can be used to detect anddistinguish hTRT RNA variants.

Another suitable method entails PCR amplification (or the equivalent)using three primers. Analogous to the semi-competitive quantitative PCRmethod described in greater detail supra, one primer is specific to eachof the hTRT RNA species (e.g., as illustrated in Table 4) and one primeris complementary to both species (e.g., TCP1.25 (2270-2288)). An exampleof a primer specific to SEQ ID NO:1 is one that anneals within the 182nucleotide sequence (i.e., nucleotides 2345 to 2526 of SEQ ID NO:1),e.g., TCP1.73 (2465-2445). For example, a primer specific to SEQ ID NO:4(a Δ182 variant) is one that anneals at nucleotides 2358 to 2339 of SEQID NO:4 (i.e., the site corresponding to the 182 nucleotide insertion inSEQ ID NO:1). The absolute abundance of the Δ182 hTRT mRNA species orits relative abundance compared to the species encoding the full-lengthhTRT protein can be analyzed for correlation to cell state (e.g.,capacity for indefinite proliferation). It will be appreciated thatnumerous other primers or amplification or detection methods can beselected based on the present disclosure.

TABLE 4 ILLUSTRATIVE PRIMERS

Δ182 species (e.g., SEQ ID NO:4) specific primer:

5′-GGCACTGGACGTAGGACGTG-3 (SEQ ID NO:550)

hTRT (SEQ ID NO:1) specific primer (TCP1.73):

5′-CACTGCTGGCCTCATTCAGGG-3 (SEQ ID NO:445)

Common (forward) primer (TCP1.25):

5′-TACTGCGTGCGTCGGTATG-3′ (SEQ ID NO:399)

Other variant hTRT genes or gene products that can be detected includethose characterized by premature stop codons, deletions, substitutionsor insertions. Deletions can be detected by the decreased size of thegene, mRNA transcript, or cDNA. Similarly, insertions can be detected bythe increased size of the gene, mRNA transcript, or cDNA insertions anddeletions could also cause shifts in the reading frame that lead topremature stop codons or longer open reading frames. Substitutions,deletions, and insertions can also be detected by probe hybridization.Alterations can also be detected by observing changes in the size of thevariant hTRT polypeptide (e.g., by Western analysis) or by hybridizationor specific amplification as appropriate. Alternatively, mutations canbe determined by sequencing of the gene or gene product according tostandard methods. In addition, and as noted above, amplification assaysand hybridization probes can be selected to target particularabnormalities specifically. For example, nucleic acid probes oramplification primers can be selected that specifically hybridize to oramplify, respectively, the region encompassing the deletion,substitution, or insertion. Where the hTRT gene harbors such a mutation,the probe will either (1) fail to hybridize or the amplificationreaction will fail to provide specific amplification or cause a changein the size of the amplification product or hybridization signal; or (2)the probe or amplification reaction encompasses the entire deletion oreither end of the deletion (deletion junction); or (3) similarly, probesand amplification primers can be selected that specifically target pointmutations or insertions.

5) Detection of Mutant hTRT Alleles

Mutations in the hTRT gene can be responsible for disease initiation orcan contribute to a disease condition. Alterations of the genomic DNA ofhTRT can affect levels of gene transcription, change amino acid residuesin the hTRT protein, cause truncated hTRT polypeptides to be produced,alter pre-mRNA processing pathways (which can alter hTRT mRNA levels),and cause other consequences as well.

Alterations of genomic DNA in non-hTRT loci can also affect expressionof hTRT or telomerase by altering the enzymes or cellular processes thatare responsible for regulating hTRT, hTR, and telomerase-associatedprotein expression and processing and RNP assembly and transport.Alterations which affect hTRT expression, processing, or RNP assemblycould be important for cancer progression, for diseases of aging, forDNA damage diseases, and others.

Detection of mutations in hTRT mRNA or its gene and gene controlelements can be accomplished in accordance with the methods herein inmultiple ways. Illustrative examples include the following: A techniquetermed primer screening can be employed; PCR primers are designed whose3′ termini anneal to nucleotides in a sample DNA (or RNA) that arepossibly mutated. If the DNA (or RNA) is amplified by the primers, thenthe 3′ termini matched the nucleotides in the gene; if the DNA is notamplified, then one or both termini did not match the nucleotides in thegene, indicating a mutation was present. Similar primer design can beused to assay for point mutations using the Ligase Chain Reaction (LCR,described supra). Restriction fragment length polymorphism, RFLP(Pourzand, C., Cerutti, P. (1993) Mutat. Res 288: 113-121), is anothertechnique that can be applied in the present method. A Southern blot ofhuman genomic DNA digested with various restriction enzymes is probedwith an hTRT specific probe. Differences in the fragment number or sizesbetween the sample and a control indicate an alteration of theexperimental sample, usually an insertion or deletion. Single strandconformation polymorphism, SSCP (Orrita, M., et al. (1989) PNAS USA86:2766-70), is another technique that can be applied in the presentmethod. SSCP is based on the differential migration of denaturedwild-type and mutant single-stranded DNA (usually generated by PCR).Single-stranded DNA will take on a three-dimensional conformation thatis sequence-specific. Sequence differences as small as a single basechange can result in a mobility shift on a nondenaturing gel. SSCP isone of the most widely used mutation screening methods because of itssimplicity. Denaturing Gradient Gel Electrophoresis, DGGE (Myers, R. M.,Maniatis, T. and Lerman, L., (1987) Methods in Enzymology, 155:501-527), is another technique that can be applied in the presentmethod. DGGE identifies mutations based on the melting behavior ofdouble-stranded DNA. Specialized denaturing electrophoresis equipment isutilized to observe the melting profile of experimental and controlDNAs: a DNA containing a mutation will have a different mobilitycompared to the control in these gel systems. The examples discussedillustrate commonly employed methodology; many other techniques existwhich are known by those skilled in the art and can be applied inaccordance with the teachings herein.

F. Karyotype Analysis

The present invention further provides methods and reagents forkaryotype or other chromosomal analysis using hTRT-sequence probesand/or detecting or locating hTRT gene sequences in chromosomes from ahuman patient, human cell line, or non-human cell. In one embodiment,amplification (i.e., change in copy number), deletion (i.e., partialdeletion), insertion, substitution, or changes in the chromosomallocation (e.g., translocation) of an hTRT gene may be correlated withthe presence of a pathological condition or a predisposition todeveloping a pathological condition (e.g., cancer).

It has been determined by the present inventors that, in normal humancells, the hTRT gene maps close to the telomere of chromosome 5p (seeExample 5, infra). The closest STS marker is D5S678 (see FIG. 8). Thelocation can be used to identify markers that are closely linked to thehTRT gene. The markers can be used to identify YACs, STSs, cosmids,BACs, lambda or P1 phage, or other clones which contain hTRT genomicsequences or control elements. The markers or the gene location can beused to scan human tissue samples for alterations in the normal hTRTgene location, organization or sequence that is associated with theoccurrence of a type of cancer or disease. This information can be usedin a diagnostic or prognostic manner for the disease or cancer involved.Moreover, the nature of any alterations to the hTRT gene can beinformative as to the nature by which cells become immortal. Forinstance, a translocation event could indicate that activation of hTRTexpression occurs in some cases by replacing the hTRT promoter withanother promoter which directs hTRT transcription in an inappropriatemanner. Methods and reagents of the invention of this type can be usedto inhibit hTRT activation. The location may also be useful fordetermining the nature of hTRT gene repression in normal somatic cells,for instance, whether the location is part of non-expressingheterochromatin. Nuclease hypersensitivity assays for distinguishingheterochromatin and euchromatin are described, for example, in Wu etal., 1979, Cell 16:797; Groudine and Weintraub, 1982, Cell 30:131 Grossand Garrard, 1988, Ann. Rev. Biochem. 57:159.

In one embodiment, alterations to the hTRT gene are identified bykaryotype analysis, using any of a variety of methods known in the art.One useful technique is in situ hybridization (ISH). Typically, when insitu hybridization techniques are used for karyotype analysis, adetectable or detectably-labeled probe is hybridized to a chromosomalsample in situ to locate an hTRT gene sequence. Generally, ISH comprisesone or more of the following steps: (1) fixation of the tissue, cell orother biological structure to be analyzed; (2) prehybridizationtreatment of the biological structure to increase accessibility oftarget DNA (e.g., denaturation with heat or alkali), and to reducenonspecific binding (e.g., by blocking the hybridization capacity ofrepetitive sequences, e.g., using human genomic DNA); (3) hybridizationof one or more nucleic acid probes (e.g., conventional nucleic acids,PNAs, or probes containing other nucleic acid analogs) to the nucleicacid in the biological structure or tissue; (4) posthybridization washesto remove nucleic acid fragments not bound in the hybridization; and,(5) detection of the hybridized nucleic acid fragments. The reagentsused in each of these steps and conditions for their use vary dependingon the particular application. It will be appreciated that these stepscan be modified in a variety of ways well known to those of skill in theart.

In one embodiment of ISH, the hTRT probe is labeled with a fluorescentlabel (fluorescent in situ hybridization; “FISH”). Typically, it isdesirable to use dual color fluorescent in situ hybridization, in whichtwo probes are utilized, each labeled by a different fluorescent dye. Atest probe that hybridizes to the hTRT sequence of interest is labeledwith one dye, and a control probe that hybridizes to a different regionis labeled with a second dye. A nucleic acid that hybridizes to a stableportion of the chromosome of interest, such as the centromere region,can be used as the control probe. In this way, one can account fordifferences between efficiency of hybridization from sample to sample.

The ISH methods for detecting chromosomal abnormalities (e.g., FISH) canbe performed on nanogram quantities of the subject nucleic acids.Paraffin embedded normal tissue or tumor sections can be used, as canfresh or frozen material, tissues, or sections. Because FISH can beapplied to limited material, touch preparations prepared from unculturedprimary tumors can also be used (see, e.g., Kallioniemi et al., 1992,Cytogenet. Cell Genet. 60:190). For instance, small biopsy tissuesamples from tumors can be used for touch preparations (see, e.g.,Kallioniemi et al., supra). Small numbers of cells obtained fromaspiration biopsy or cells in bodily fluids (e.g., blood, urine, sputumand the like) can also be analyzed. For prenatal diagnosis, appropriatesamples will include amniotic fluid, maternal blood, and the like.Useful hybridization protocols applicable to the methods and reagentsdisclosed here are described in Pinkel et al., 1988, Proc. Natl. Acad.Sci. USA, 85:9138; EPO Pub. No. 430,402; Choo, ed., Methods in MolecularBiology Vol. 33: In Situ Hybridization Protocols, Humana Press, Totowa,N.J., (1994); and Kallioniemi et al., supra.

Other techniques useful for karyotype analysis include, for example,techniques such as quantitative Southern blotting, quantitative PCR, orcomparative genomic hybridization (Kallioniemi et al., 1992, Science,258:818), using the hTRT probes and primers of the invention which maybe used to identify amplification, deletion, insertion, substitution orother rearrangement of hTRT sequences in chromosomes in a biologicalsample.

G. TRT Polypeptide Assays

1) Generally

The present invention provides methods and reagents for detecting andquantitating hTRT polypeptides. These methods include analyticalbiochemical methods such as electrophoresis, mass spectroscopy, gelshift, capillary electrophoresis, chromatographic methods such as sizeexclusion chromatography, high performance liquid chromatography (HPLC),thin layer chromatography (TLC), hyperdiffusion chromatography, and thelike, or various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, western blotting, mass spectrometry, andothers described below and apparent to those of skill in the art uponreview of this disclosure.

2) Electrophoretic Assays

In one embodiment, the hTRT polypeptides are detected in anelectrophoretic protein separation; in one aspect, a two-dimensionalelectrophoresis system is employed. Means of detecting proteins usingelectrophoretic techniques are well known to those of skill in the art(see generally, R. Scopes (1982) Protein Purification, Springer-Verlag,N.Y.; Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to ProteinPurification, Academic Press, Inc., N.Y.).

In a related embodiment, a mobility shift assay (see, e.g., Ausubel etal., supra) is used. For example, labeled-hTR will associate with hTRTand migrate with altered mobility upon electrophoresis in anondenaturing polyacrylamide gel or the like. Thus, for example, if an(optionally labeled) hTR probe or a (optionally labeled) telomeraseprimer is mixed with a sample containing hTRT, or coexpressed with hTRT(e.g., in a cell-free expression system) the presence of hTRT protein(or a polynucleotide encoding hTRT) in the sample will result in adetectable alteration of hTR mobility.

3) Immunoassays

a) Generally

The present invention also provides methods for detection of hTRTpolypeptides employing one or more antibody reagents of the invention(i.e., immunoassays). As used herein, an immunoassay is an assay thatutilizes an antibody (as broadly defined herein and specificallyincludes fragments, chimeras and other binding agents) that specificallybinds an hTRT polypeptide or epitope. Antibodies of the invention may bemade by a variety of means well known to those of skill in the art,e.g., as described supra.

A number of well established immunological binding assay formatssuitable for the practice of the invention are known (see, e.g., U.S.Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). See, e.g.,Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Asai, ed.Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7thEdition, Stites & Terr, eds. (1991); Harlow and Lane, supra [e.g.,Chapter 14], and Ausubel et al., suptra, [e.g., Chapter 11], each ofwhich is incorporated by reference in its entirety and for all purposes.Typically, immunological binding assays (or immunoassays) utilize a“capture agent” to specifically bind to and, often, immobilize theanalyte. In one embodiment, the capture agent is a moiety thatspecifically binds to an hTRT polypeptide or subsequence, such as ananti-hTRT antibody. In an alternative embodiment, the capture agent maybind an hTRT-associated protein or RNA under conditions in which thehTRT-associated molecule remains bound to the hTRT (such that if thehTRT-associated molecule is immobilized the hTRT protein is similarlyimmobilized). It will be understood that in assays in which anhTRT-associated molecule is captured the associated hTRT protein willusually be present and so can be detected, e.g., using an anti-hTRTantibody or the like. Immunoassays for detecting protein complexes areknown in the art (see, e.g., Harlow and Lane, supra, at page 583).

Usually the hTRT gene product being assayed is detected directly orindirectly using a detectable label. The particular label or detectablegroup used in the assay is usually not a critical aspect of theinvention, so long as it does not significantly interfere with thespecific binding of the antibody or antibodies used in the assay. Thelabel may be covalently attached to the capture agent (e.g., an anti-TRTantibody), or may be attached to a third moiety, such as anotherantibody, that specifically binds to, e.g.,: the hTRT polypeptide (at adifferent epitope than recognized by the capture agent), the captureagent (e.g., an anti-(first antibody) immunoglobulin); an anti-TRTantibody; an antibody that binds an anti-TRT antibody; or, anantibody/telomerase complex (e.g., via binding to an associated moleculesuch as a telomerase-associated protein). Other proteins capable ofbinding an antibody used in the assay, such as protein A or protein G,may also be labeled. In some embodiments, it will be useful to use morethan one labeled molecule (i.e., ones that can be distinguished from oneanother). In addition, when the target bound (e.g., immobilized) by thecapture agent (e.g., anti-hTRT antibody) is a complex (i.e., a complexof hTRT and a TRT-associated protein, hTR, or other TRT associatedmolecule), a labeled antibody that recognizes the protein or RNAassociated with the hTRT protein can be used. When the complex is aprotein-nucleic acid complex (e.g., TRT-hTR), the reporter molecule canbe a polynucleotide or other molecule (e.g., enzyme) that recognizes theRNA component of the complex.

Some immunoassay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, the components do not need to be labeled, and the presence ofthe target antibody can be detected by simple visual inspection.

b) Non-Competitive Assay Formats

The present invention provides methods and reagents for competitive andnoncompetitive immunoassays for detecting hTRT polypeptides.Noncompetitive immunoassays are assays in which the amount of capturedanalyte (in this case hTRT) is directly measured. One such assay is atwo-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on the hTRT protein. See, e.g.,Maddox et al., 1983, J. Exp. Med., 158:1211 for background information.In one preferred “sandwich” assay, the capture agent (e.g., an anti-TRTantibody) is bound directly to a solid substrate where it isimmobilized. These immobilized antibodies then capture any hTRT proteinpresent in the test sample. The hTRT thus immobilized can then belabeled, i.e., by binding to a second anti-hTRT antibody bearing alabel. Alternatively, the second anti-hTRT antibody may lack a label,but be bound by a labeled third antibody specific to antibodies of thespecies from which the second antibody is derived. The second antibodyalternatively can be modified with a detectable moiety, such as biotin,to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

c) Competitive Assay Formats

In competitive assays, the amount of hTRT protein present in the sampleis measured indirectly by measuring the amount of an added (exogenous)hTRT displaced (or competed away) from a capture agent (e.g., anti-TRTantibody) by the hTRT protein present in the sample. In one competitiveassay, a known amount of labeled hTRT protein is added to the sample andthe sample is then contacted with a capture agent (e.g., an antibodythat specifically binds hTRT protein). The amount of exogenous (labeled)hTRT protein bound to the antibody is inversely proportional to theconcentration of hTRT protein present in the sample. In one embodiment,the antibody is immobilized on a solid substrate. The amount of hTRTprotein bound to the antibody may be determined either by measuring theamount of hTRT protein present in a TRT/antibody complex, oralternatively by measuring the amount of remaining uncomplexed TRTprotein. The amount of hTRT protein may be detected by providing alabeled hTRT molecule.

A hapten inhibition assay is another example of a competitive assay. Inthis assay hTRT protein is immobilized on a solid substrate. A knownamount of anti-TRT antibody is added to the sample, and the sample isthen contacted with the immobilized hTRT protein. In this case, theamount of anti-TRT antibody bound to the immobilized hTRT protein isinversely proportional to the amount of hTRT protein present in thesample. The amount of immobilized antibody may be detected by detectingeither the immobilized fraction of antibody or the fraction of theantibody that remains in solution. In this aspect, detection may bedirect, where the antibody is labeled, or indirect where the label isbound to a molecule that specifically binds to the antibody as describedabove.

d) Other Assay Formats

The invention also provides reagents and methods for detecting andquantifying the presence of hTRT in the sample by using an immunoblot(Western blot) format. In this format, hTRT polypeptides in a sample areseparated from other sample components by gel electrophoresis (e.g., onthe basis of molecular weight), the separated proteins are transferredto a suitable solid support (such as a nitrocellulose filter, a nylonfilter, derivatized nylon filter, or the like), and the support isincubated with anti-TRT antibodies of the invention. The anti-TRTantibodies specifically bind to hTRT or other TRT on the solid support.These antibodies may be directly labeled or alternatively may besubsequently detected using labeled antibodies (e.g., labeled sheepanti-mouse antibodies) or other labeling reagents that specifically bindto the anti-TRT antibody.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals canthen be detected according to standard techniques (see, Monroe et al.,1986, Amer. Clin. Prod. Rev. 5:34).

As noted supra, assay formats using FACS (and equivalent instruments ormethods) have advantages when measuring hTRT gene products in aheterogeneous sample (such as a biopsy sample containing both normal andmalignant cells).

e) Substrates, Solid Supports, Membranes, Filters

As noted supra, depending upon the assay, various components, includingthe antigen, target antibody, or anti-hTRT antibody, may be bound to asolid surface or support (i.e., a substrate, membrane, or filter paper).Many methods for immobilizing biomolecules to a variety of solidsurfaces are known in the art. For instance, the solid surface may be amembrane (e.g., nitrocellulose), a microtiter dish (e.g., PVC,polypropylene, or polystyrene), a test tube (glass or plastic), adipstick (e.g. glass, PVC, polypropylene, polystyrene, latex, and thelike), a microcentrifuge tube, or a glass or plastic bead. The desiredcomponent may be covalently bound or noncovalently attached throughnonspecific bonding.

A wide variety of organic and inorganic polymers, both natural andsynthetic may be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidenedifluoride (PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials which may beemployed, include paper, glasses, ceramics, metals, metalloids,semiconductive materials, cements or the like. In addition, substancesthat form gels, such as proteins (e.g., gelatins), lipopolysaccharides,silicates, agarose and polyacrylamides can be used. Polymers which formseveral aqueous phases, such as dextrans, polyalkylene glycols orsurfactants, such as phospholipids, long chain (12-24 carbon atoms)alkyl ammonium salts and the like are also suitable. Where the solidsurface is porous, various pore sizes may be employed depending upon thenature of the system.

In preparing the surface, a plurality of different materials may beemployed, particularly as laminates, to obtain various properties. Forexample, protein coatings, such as gelatin can be used to avoidnon-specific binding, simplify covalent conjugation, enhance signaldetection or the like.

If covalent bonding between a compound and the surface is desired, thesurface will usually be polyfunctional or be capable of beingpolyfunctionalized. Functional groups which may be present on thesurface and used for linking can include carboxylic acids, aldehydes,amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercaptogroups and the like. The manner of linking a wide variety of compoundsto various surfaces is well known and is amply illustrated in theliterature. See, for example, Immobilized Enzymes, Ichiro Chibata,Halsted Press, New York, 1978, and Cuatrecasas (1970) J. Biol. Chem. 2453059).

In addition to covalent bonding, various methods for noncovalentlybinding an assay component can be used. Noncovalent binding is typicallynonspecific absorption of a compound to the surface.

One of skill in the art will appreciate that it is often desirable toreduce non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this involves coating thesubstrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk sometimes preferred.Alternatively, the surface is designed such that it nonspecificallybinds one component but does not significantly bind another. Forexample, a surface bearing a lectin such as Concanavalin A will bind acarbohydrate containing compound but not a labeled protein that lacksglycosylation. Various solid surfaces for use in noncovalent attachmentof assay components are reviewed in U.S. Pat. Nos. 4,447,576 and4,254,082.

H) Assays for Anti-TRT Antibodies

The present invention also provides reagents and assays for detectinghTRT-specific immunoglobulins. In one embodiment, immobilized hTRT(e.g., recombinant hTRT bound to a microassay plate well) is incubatedwith serum from a patient under conditions in which anti-hTRTantibodies, if present, bind the immobilized hTRT. After washing toremove nonspecifically bound immunoglobulin, bound serum antibodies canbe detected, if they are present, by adding detectably labeledanti-(human Ig) antibodies (alternative embodiments and variations arewell known to those of skill in the art; see, e.g., Harlow, supra, atCh. 14). These assays are useful for detecting anti-hTRT antibodies inany source including animal or human serum or a carrier such as saline.In one embodiment, the assays are used to detect or monitor an immuneresponse to hTRT proteins in a patient, particularly an autoimmune(e.g., anti-telomerase) response. Anti-hTRT antibodies may be present inthe serum or other tissues or fluids from a patient suffering from anautoimmune disease or other condition.

I) Assay Combinations

The diagnostic and prognostic assays described herein can be carried outin various combinations and can also be carried out in conjunction withother diagnostic or prognostic tests. For example, when the presentmethods are used to detect the presence of cancer cells in patientsample, the presence of hTRT can be used to determine the stage of thedisease, whether a particular tumor is likely to invade adjoining tissueor metastasize to a distant location, and whether a recurrence of thecancer is likely. Tests that may provide additional information includemicroscopic analysis of biopsy samples, detection of antigens (e.g.,cell-surface markers) associated with tumorigenicity (e.g., usinghistocytochemistry, FACS, or the like), imaging methods (e.g., uponadministration to a patient of labeled anti-tumor antibodies),telomerase activity assays, telomere length assays, hTR assays, or thelike. Such combination tests can provide useful information regardingthe progression of a disease.

It will also be recognized that combinations of assays can provideuseful information. For example, and as noted above, assays for hTRTmRNA can be combined with assays for hTR (human telomerase RNA) ortelomerase activity (i.e., TRAP) assays to provide information abouttelomerase assembly and function.

J) Kits

The present invention also provides kits useful for the screening,monitoring, diagnosis and prognosis of patients with atelomerase-related condition, or for determination of the level ofexpression of hTRT in cells or cell lines. The kits include one or morereagents for determining the presence or absence of an hTRT gene product(RNA or protein) or for quantifying expression of the hTRT gene.Preferred reagents include nucleic acid primers and probes thatspecifically bind to the hTRT gene, RNA, cDNA, or portions thereof,along with proteins, peptides, antibodies, and control primers, probes,oligonucleotides, proteins, peptides and antibodies. Other materials,including enzymes (e.g., reverse transcriptases, DNA polymerases,ligases), buffers, reagents (labels, dNTPs), may be included.

The kits may include alternatively, or in combination with any of theother components described herein, an antibody that specifically bindsto hTRT polypeptides or subsequences thereof. The antibody can bemonoclonal or polyclonal. The antibody can be conjugated to anothermoiety such as a label and/or it can be immobilized on a solid support(substrate). The kit(s) may also contain a second antibody for detectionof hTRT polypeptide/antibody complexes or for detection of hybridizednucleic acid probes, as well as one or more hTRT peptides or proteinsfor use as control or other reagents.

The antibody or hybridization probe may be free or immobilized on asolid support such as a test tube, a microtiter plate, a dipstick andthe like. The kit may also contain instructional materials teaching theuse of the antibody or hybridization probe in an assay for the detectionof TRT. The kit may contain appropriate reagents for detection oflabels, or for labeling positive and negative controls, washingsolutions, dilution buffers and the like.

In one embodiment, the kit includes a primer pair for amplifying hTRTmRNA. Such a kit may also include a probe for hTRT amplified DNA and/ora polymerase, buffer, dNTPs, and the like. In another, the kit comprisesa probe, optionally a labeled probe. In another, the kit comprises anantibody.

X. Identification of Modulators of Telomerase Activity

A. Generally

The invention provides compounds and treatments that modulate theactivity or expression of a telomerase or telomerase component (e.g.,hTRT protein). The invention also provides assays and screening methods(including high-throughput screens) for identification of compounds andtreatments that modulate telomerase activity or expression. Thesemodulators of telomerase activity and expression (hereinafter referredto as “modulators”) include telomerase agonists (which increasetelomerase activity and/or expression) and telomerase antagonists (whichdecrease telomerase activity and/or expression).

The modulators of the invention have a wide variety of uses. Forexample, it is contemplated that telomerase modulators will be effectivetherapeutic agents for treatment of human diseases. Screening foragonist activity and transcriptional or translational activatorsprovides for compositions that increase telomerase activity in a cell(including a telomere dependent replicative capacity, or a “partial”telomerase activity). Such agonist compositions provide for methods ofimmortalizing otherwise normal untransformed cells, including cellswhich can express useful proteins. Such agonists can also provide formethods of controlling cellular senescence. Conversely, screening forantagonist activity provides for compositions that decrease telomeredependent replicative capacity, thereby mortalizing otherwise immortalcells, such as cancer cells. Screening for antagonist activity providesfor compositions that decrease telomerase activity, thereby preventingunlimited cell division of cells exhibiting unregulated cell growth,such as cancer cells. Illustrative diseases and conditions that may betreated using modulators are listed herein, e.g., in Sections VIII andIX, supra. In general, the modulators of the invention can be usedwhenever it is desired to increase or decrease a telomerase activity ina cell or organism. Thus, in addition to use in treatment of disease, amodulator that increases hTRT expression levels can be used to produce acultured human cell line having properties as generally described inSection VIII, supra, and various other uses that will be apparent to oneof skill.

A compound or treatment modulates “expression” of telomerase or atelomerase component when administration of the compound or treatmentchanges the rate or level of transcription of the gene encoding atelomerase component (e.g., the gene encoding hTRT mRNA), affectsstability or post-transcriptional processing of RNA encoding atelomerase component (e.g., transport, splicing, polyadenylation, orother modification), affects translation, stability, post-translationalprocessing or modification of an encoded protein (e.g., hTRT), orotherwise changes the level of functional (e.g., catalytically active)telomerase RNP. A compound or treatment affects a telomerase “activity”when administration of the compound or treatment changes a telomeraseactivity such as any activity described in Section IV(B), supra (e.g.,including processive or non-processive telomerase catalytic activity;telomerase processivity; conventional reverse transcriptase activity;nucleolytic activity; primer or substrate binding activity; dNTP bindingactivity; RNA binding activity; telomerase RNP assembly; and proteinbinding activity). It will be appreciated that there is not necessarilya sharp delineation between changes in “activity” and changes in“expression,” and that these terms are used for ease of discussion andnot for limitation. It will also be appreciated that the modulators ofthe invention should specifically affect telomerase activity orexpression (e.g., without generally changing the expression ofhousekeeping proteins such as actin) rather than, for example, reducingexpression of a telomerase component by nonspecific poisoning of atarget cell.

B. Assays for Identification of Telomerase Modulators

The invention provides methods and reagents to screen for compositionsor compounds capable of affecting expression of a telomerase ortelomerase component, capable of modifying the DNA replicative capacityof telomerase, or otherwise modifying the ability of the telomeraseenzyme and TRT protein to synthesize telomeric DNA (“full activity”).The invention also provides screens for modulators of any or all ofhTRT's “partial activities.” Thus, the present invention provides assaysthat can be used to screen for agents that increase the activity oftelomerase, for example, by causing hTRT protein or telomerase to beexpressed in a cell in which it normally is not expressed or byincreasing telomerase activity levels in telomerase positive cells.

Telomerase or telomerase subunit proteins or their catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening therapeutic compounds in any of a variety of drug screeningtechniques. The fragment employed in such a test may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweentelomerase or the subunit protein and the agent being tested, may bemeasured.

In various embodiments, the invention includes methods for screening forantagonists that: bind to the enzyme's active site; inhibit theassociation of its RNA moiety, telomerase-associated proteins,nucleotides, or telomeric DNA to telomerase or hTRT protein; promote thedisassociation of the enzyme complex; interfere with transcription ofthe telomerase RNA moiety (e.g., hTR); or inhibit any of the “partialactivities” described herein. The invention provides methods forscreening for compositions that inhibit the association of nucleic acidand/or telomerase-associated compositions with hTRT, such as theassociation of hTR with hTRT or the association of hTRT with the humanhomologs of p80 or p95 or another associated protein, or association ofhTRT with a telomere or a nucleotide; screening for compositions thatpromote the disassociation or promote the association (i.e., assembly)of the enzyme complex, such as an antibody directed to hTR or hTRT;screening for agents that effect the processivity of the enzyme; andscreening for nucleic acids and other compositions that bind totelomerase, such as a nucleic acid complementary to hTR. The inventionfurther contemplates screening for compositions that increase ordecrease the transcription of the hTRT gene and/or translation of thehTRT gene product. The invention also contemplates a method of screeningfor telomerase modulators in animals, in one embodiment, byreconstituting a telomerase activity, or an anti-telomerase activity, inan animal, such as a transgenic animal. The invention provides for invivo assays systems that include “knockout” models, in which one orseveral units of the endogenous telomerase, telomerase RNA moiety and/ortelomerase-associated proteins have been deleted or inhibited. Theendogenous telomerase activity, full or partial, can remain or beabsent. In one embodiment, an exogenous telomerase activity, full orpartial, is reconstituted.

In one embodiment of the invention, a variety of partial activitytelomerase assays are provided to identify a variety of differentclasses of modulators of telomerase activity. The “partial activity”assays of the invention allow identification of classes of telomeraseactivity modulators that might otherwise not be detected in a “fullactivity” telomerase assay. One partial activity assay involves thenon-processive activity of TRT and telomerase. The processive nature oftelomerase is described by Morin (1989) Cell 59:521-529; see also Prowse(1993) “Identification of a nonprocessive telomerase activity from mousecells” Proc. Natl. Acad. Sci. USA 90:1493-1497. Another partial activityassay of the invention exploits the “reverse-transcriptase-like”activity of telomerase. In these assays, one assays the reversetranscriptase activity of the hTRT protein. See Lingner (1997) “Reversetranscriptase motifs in the catalytic subunit of telomerase” Science276:561-567. Another partial activity assay of the invention exploitsthe “nucleolytic activity” of hTRT and telomerase, involving theenzyme's removing of at least one nucleotide, typically guanosine, fromthe 3′ strand of a primer. This nucleolytic activity has been observedin Tetrahymena telomerase by Collins (1993) “Tetrahymena telomerasecatalyzes nucleolytic cleavage and nonprocessive elongation” Genes Dev7:1364-1376. Another partial activity assay of the invention involvesanalyzing hTRT's and telomerase's ability to bind nucleotides as part ofits enzymatic processive DNA polymerization activity. Another partialactivity assay of the invention involves analyzing hTRT's ortelomerase's ability to bind its RNA moiety, i.e., hTR for human cells,used as a template for telomere synthesis. Additional partial activityassays of the invention involve analyzing hTRT's and telomerase'sability to bind chromosomes in vivo, or to bind oligonucleotide primersin vitro or in reconstituted systems, or to bind proteins associatedwith chromosomal structure (see, for an example of such a protein,Harrington (1995) J Biol Chem 270: 8893-8901). Chromosomal structureswhich bind hTRT include, for example, telomeric repeat DNA, telomereproteins, histones, nuclear matrix protein, cell division/ cell cyclecontrol proteins and the like.

In one embodiment, an assay for identification of modulators comprisescontacting one or more cells (i.e., “test cells”) with a test compound,and determining whether the test compound affects expression or activityof a telomerase (or telomerase component) in the cell. Usually thisdetermination comprises comparing the activity or expression in the testcell compared to a similar cell or cells (i.e., control cells) that havenot been contacted with the test compound. Alternatively, cell extractsmay be used in place of intact cells. In a related embodiment, the testcompound is administered to a multicellular organism (e.g., a plant oranimal). The telomerase or telomerase component may be wholly endogenousto the cell or multicellular organism (i.e., encoded by naturallyoccurring endogenous genes), or may be a recombinant cell or transgenicorganism comprising one or more recombinantly expressed telomerasecomponents (e.g., hTRT, hTR, telomerase-associated proteins), or mayhave both endogenous and recombinant components. Thus, in oneembodiment, telomerase-activity-modulators are administered to mortalcells. In another embodiment, telomerase-activity-modulators areadministered to immortal cells. For example, antagonists oftelomerase-mediated DNA replication can be identified by administeringthe putative inhibitory composition to a cell that is known to exhibitsignificant amounts of telomerase activity, such as cancer cells, andmeasuring whether a decrease in telomerase activity, telomere length, orproliferative capacity is observed, all of which are indicative of acompound with antagonist activity.

In another embodiment, a modulator is identified by monitoring a changein a telomerase activity of a ribonucleoprotein complex (RNP) comprisinga TRT (e.g., hTRT) and a template RNA (e.g., hTR), which RNP isreconstituted in vitro (e.g., as described in Example 7, infra).

In yet another embodiment, the modulator is identified by monitoring achange in expression of a TRT gene product (e.g., RNA or protein) in acell, animal, in vitro expression system, or other expression system.

In still another embodiment, the modulator is identified by changing theexpression of a reporter gene, such as that described in Example 15,whose expression is regulated, in whole or part, by a naturallyoccurring TRT regulatory element such as a promoter or enhancer. In arelated embodiment, the ability of a test compound to bind to atelomerase component (e.g., hTRT), RNA, or gene regulatory sequence(e.g., the TRT gene promoter) is assayed.

In another embodiment, the modulator is identified by observing changesin hTRT pre-mRNA processing, for example, alternatively splicedproducts, alternative poly-adenylation events, RNA cleavage, and thelike. In a related embodiment the activity of the modulator can beobserved by monitoring the production of variant hTRT polypeptides, someof which may possess dominant-negative telomerase regulation activity.

Assay formats for identification of compounds that affect expression andactivity of proteins are well known in the biotechnological andpharmaceutical industries, and numerous additional assays and variationsof the illustrative assays provided supra will be apparent to those ofskill.

Changes in telomerase activity or expression can be measured by anysuitable method. Changes in levels of expression of a telomerasecomponent (e.g., hTRT protein) or precursor (e.g., hTRT mRNA) can beassayed using methods well known to those of skill, some of which aredescribed hereinabove, e.g., in Section IX and including monitoringlevels of TRT gene products (e.g., protein and RNAs) by hybridization(e.g., using the TRT probes and primers of the invention), immunoassays(e.g., using the anti-TRT antibodies of the invention), RNAse protectionassays, amplification assays, or any other suitable detection meansdescribed herein or known in the art. Quantitating amounts of nucleicacid in a sample (e.g., evaluating levels of RNA, e.g., hTR or hTRTmRNA) is also useful in evaluating cis- or trans-transcriptionalregulators.

Similarly, changes in telomerase activity can be measured using methodssuch as those described herein (e.g., in Section IV(B), supra) or otherassays of telomerase function. Quantitation of telomerase activity, whendesired, may be carried out by any method, including those disclosedherein. Telomerase antagonists that can cause or accelerate loss oftelomeric structure can be identified by monitoring and measuring theireffect on telomerase activity in vivo, ex vivo, or in vitro, or by theireffects on telomere length (as measured or detected through staining,use of tagged hybridization probes or other means) or, simply, by theinhibition of cell division of telomerase positive cancer cells(critical shortening of telomeres leads to a phenomenon termed “crisis”or M2 senescence (Shay, 1991) Biochem. Biophys. Acta 1072:1-7), whichcancer cells have bypassed by the activation of telomerase, but which,in the absence of telomerase, will lead to their senescence or deaththrough chromosomal deletion and rearrangement). The in vivo humantelomerase activity reconstitution provides for a method of screeningfor telomerase modulators in cells or animals from any origin. Suchagonists can be identified in an activity assay of the invention,including measurements of changes in telomere length. Other examples ofassays measuring telomerase activity in cells include assays for theaccumulation or loss of telomere structure, the TRAP assay or aquantitative polymerase chain reaction assay.

In one embodiment, the assays of the invention also include a methodwhere the test compound produces a statistically significant decrease inthe activity of hTRT as measured by the incorporation of a labelednucleotide into a substrate compared to the relative amount ofincorporated label in a parallel reaction lacking the test compound,thereby determining that the test compound is a telomerase inhibitor.

The methods of the invention are amenable to adaptations from protocolsdescribed in the scientific and patent literature and known in the art.For example, when a telomerase or TRT protein of this invention is usedto identify compositions which act as modulators of telomeraseactivities, large numbers of potentially useful molecules can bescreened in a single test. The modulators can have an inhibitory(antagonist) or potentiating (agonist) effect on telomerase activity.For example, if a panel of 1,000 inhibitors is to be screened, all 1,000inhibitors can potentially be placed into one microtiter well and testedsimultaneously. If such an inhibitor is discovered, then the pool of1,000 can be subdivided into 10 pools of 100 and the process repeateduntil an individual inhibitor is identified.

In drug screening large numbers of compounds are examined for theirability to act as telomerase modulators, a process greatly acceleratedby the techniques of high throughput screening. The assays fortelomerase activity, full or partial, described herein may be adapted tobe used in a high throughput technique. Those skilled in the artappreciate that there are numerous methods for accomplishing thispurpose.

Another technique for drug screening which may be applied for highthroughput screening of compounds having suitable binding affinity tothe telomerase or telomerase protein subunit is described in detail in“Determination of Amino Acid Sequence Antigenicity” by Geysen, (Geysen,WO Application 84/03564, published on Sep. 13, 1984, incorporated hereinby reference). In summary, large numbers of different small peptide testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The peptide test compounds are reacted withfragments of telomerase or telomerase protein subunits and washed. Boundtelomerase or telomerase protein subunit is then detected by methodswell known in the art. Substantially purified telomerase or telomeraseprotein subunit can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding telomerase orsubunit protein(s) specifically compete with a test compound for bindingtelomerase or the subunit protein. Antibodies can also be used to detectthe presence of any peptide which shares one or more antigenicdeterminants with the telomerase or subunit protein.

Additional methods for identifying modulators of a telomerase activityhave been described in U.S. Pat. No. 5,645,986, which is incorporatedherein by reference. It will be appreciated that the present inventionprovides improvements to previously known methods, in part by providingreagents such as hTRT polynucleotides, probes and primers, highlypurified hTR, hTRT and telomerase, as well as anti-telomerase andanti-TRT antibodies, all of which may be used in assays, e.g., ascontrols, standards, binding or hybridization agents, or otherwise.

It will be recognized that the recombinantly produced telomerase and TRT(e.g., hTRT) of the invention will be useful in assays foridentification of modulators. The screening assay can utilize telomeraseor hTRT derived by a full or partial reconstitution of telomeraseactivity, or by an augmentation of existing activity. The assay orscreens provided by the invention can be used to test for the ability oftelomerase to synthesize telomeric DNA or to test for any one or all orof the “partial activities” of hTRT and TRTs generally, as describedabove. The assay can incorporate ex vivo modification of cells whichhave been manipulated to express telomerase with or without its RNAmoiety or associated proteins, and these can be re-implanted into ananimal, which can be used for in vivo testing. Thus, this inventionprovides in vivo assays and transgenic animals useful therein. These invivo assays systems can employ “knockout” cells, in which one or severalunits of the endogenous telomerase enzyme complex have been deleted orinhibited, as well as cells in which an exogenous or endogenoustelomerase activity is reconstituted or activated.

Telomerases and TRT proteins that have been modified in a site-specificmanner (by site-specific mutation) to modify or delete any or allfunctions of the telomerase enzyme or the TRT protein can also beemployed in the screens of the invention to discover therapeutic agents.For example, the TRT can be engineered to lose its ability to bindsubstrate DNA, to bind its RNA moiety (as hTR), to catalyze the additionof telomeric DNA, to bind deoxynucleotide substrate, to have nucleolyticactivity, to bind telomere-associated proteins or chromosomalstructures, and the like. The resulting “mutant proteins” or “muteens”can be used to identify compounds that specifically modulate one,several, or all functions or activities of the TRT protein ortelomerase.

C. Exemplary Telomerase Modulators

1) Generally

The test compounds referred to supra may be any of a large variety ofcompounds, both naturally occurring and synthetic, organic andinorganic, and including polymers (e.g., oligopeptides, polypeptides,oligonucleotides, and polynucleotides), small molecules, antibodies (asbroadly defined herein), sugars, fatty acids, nucleotides and nucleotideanalogs, analogs of naturally occurring structures (e.g., peptidemimetics, nucleic acid analogs, and the like), and numerous othercompounds.

The invention provides modulators of all types, without limitation toany particular mechanism of action. For illustrative purposes, examplesof modulators include compounds or treatments that:

(i) bind to the hTRT polypeptide (e.g., the active site of the enzyme)or other telomerase component, and affect a telomerase activity;

(ii) inhibit or promote association, or inhibit or promotedisassociation, of a telomerase component (e.g., hTRT or the hTRT-hTRRNP) with or from a telomerase-associated protein (e.g., including thosedescribed in Section IV(D), supra);

(iii) inhibit or promote association, or inhibit or promotedisassociation, of telomerase polypeptides (e.g., hTRT) with or from atelomerase RNA (e.g., hTR);

(iv) inhibit or promote association, or inhibit or promotedisassociation, of telomerase polypeptides (e.g., hTRT) with or fromchromosomes (e.g., telomeres) or chromosomal DNA (e.g. telomeric DNA);

(v) increase or decrease expression of a telomerase component geneproduct (e.g., products of the hTRT gene), including change the rate orlevel of transcription of the TRT gene, or translation, transport orstability of a gene product, or the like, by binding to the gene or geneproduct (e.g., by interacting with a factor (e.g., a transcriptionregulatory protein) that affects transcription of the hTRT gene oranother telomerase component).

2) Peptide Modulators

Potential modulators of telomerase activity also include peptides (e.g.,inhibitory (antagonist) and activator (agonist) peptide modulators). Forexample, oligopeptides with randomly generated sequences can be screenedto discover peptide modulators (agonists or inhibitors) of telomeraseactivity. Such peptides can be used directly as drugs or to find theorientation or position of a functional group that can inhibittelomerase activity that, in turn, leads to design and testing of asmall molecule inhibitor, or becomes the backbone for chemicalmodifications that increase pharmacological utility. Peptides can bestructural mimetics, and one can use molecular modeling programs todesign mimetics based on the characteristic secondary structure and/ortertiary structure of telomerase enzyme and hTRT protein. Suchstructural mimetics can also be used therapeutically, in vivo, asmodulators of telomerase activity (agonists and antagonists). Structuralmimetics can also be used as immunogens to elicit anti-telomerase oranti-TRT protein antibodies.

3) Inhibitory Natural Compounds as Modulators of Telomerase Activity

In addition, a large number of potentially useful activity-modifyingcompounds can be screened in extracts from natural products as a sourcematerial. Sources of such extracts can be from a large number of speciesof fungi, actinomyces, algae, insects, protozoa, plants, and bacteria.Those extracts showing inhibitory activity can then be analyzed toisolate the active molecule. See for example, Turner (1996) J.Ethnopharmacol 51(1-3):39-43; Suh (1995) Anticancer Res. 15:233-239.

4) Inhibitory Oligonucleotides

One particularly useful set of inhibitors provided by the presentinvention includes oligonucleotides which are able to either bind mRNAencoding hTRT protein or to the hTRT gene, in either case preventing orinhibiting the production of functional hTRT protein. Otheroligonucleotides of the invention interact with telomerase's RNA moiety,such as hTR, or are able to prevent binding of telomerase or hTRT to itsDNA target, or one telomerase component to another, or to a substrate.Such oligonucleotides can also bind the telomerase enzyme, hTRT protein,or both protein and RNA and inhibit a partial activity as describedabove (such as its processive activity, its reverse transcriptaseactivity, its nucleolytic activity, and the like). The association canbe through sequence specific hybridization to another nucleic acid or bygeneral binding, as in an aptamer, or both.

Telomerase activity can be inhibited by targeting the hTRT mRNA withantisense oligonucleotides capable of binding the hTRT mRNA.

Another useful class of inhibitors includes oligonucleotides which causeinactivation or cleavage of hTRT mRNA or hTR. That is, theoligonucleotide is chemically modified, or has enzyme activity, whichcauses such cleavage, such as is the case for a ribozyme, anEDTA-tethered oligonucleotide, or a covalently bound oligonucleotide,such as a psoralen or other cross-linking reagent bound oligonucleotide.As noted above, one may screen a pool of many different sucholigonucleotides for those with the desired activity.

Another useful class of inhibitors includes oligonucleotides which bindpolypeptides. Double- or single-stranded DNA or double- orsingle-stranded RNA molecules that bind to specific polypeptides targetsare called “aptamers.” The specific oligonucleotide-polypeptideassociation may be mediated by electrostatic interactions. For example,aptamers specifically bind to anion-binding exosites on thrombin, whichphysiologically binds to the polyanionic heparin (Bock (1992) Nature355:564-566). Because hTRT protein binds both hTR and its DNA substrate,and because the present invention provides hTRT and other TRT proteinsin purified form in large quantities, those of skill in the art canreadily screen for TRT-binding aptamers using the methods of theinvention.

Oligonucleotides (e.g., RNA oligonucleotides) that bind telomerase,hTRT, hTR, or portions thereof, can be generated using the techniques ofSELEX (Tuerk, 1997, Methods Mol Biol 67, 2190). In this technique a verylarge pool (106-109) of random sequence nucleic acids is bound to thetarget (e.g. hTRT) using conditions that cause a large amount ofdiscrimination between molecules with high affinity and low affinity forbinding the target. The bound molecules are separated from unbound, andthe bound molecules are amplified by virtue of a specific nucleic acidsequence included at their termini and suitable amplification reagents.This process is reiterated several times until a relatively small numberof molecules remain that possess high binding affinity for the target.These molecules can then be tested for their ability to modulatetelomerase activity as described herein.

Antagonists of telomerase-mediated DNA replication can also be based oninhibition of hTR (Norton (1996) Nature Biotechnology 14:615-619)through complementary sequence recognition or cleavage, as throughribozymes.

The inhibitory oligonucleotides of the invention can be transferred intothe cell using a variety of techniques well known in the art. Forexample, oligonucleotides can be delivered into the cytoplasm withoutspecific modification. Alternatively, they can be delivered by the useof liposomes which fuse with the cellular membrane or are endocytosed,i.e., by employing ligands attached to the liposome or directly to theoligonucleotide, that bind to surface membrane protein receptors of thecell resulting in endocytosis. Alternatively, the cells may bepermeabilized to enhance transport of the oligonucleotides into thecell, without injuring the host cells. One can use a DNA bindingprotein, e.g., HBGF-1, known to transport an oligonucleotide into acell.

5) Inhibitory Ribozymes

Ribozymes act by binding to a target RNA through the target RNA bindingportion of a ribozyme which is held in close proximity to an enzymaticportion of the ribozyme that cleaves the target RNA. Thus, the ribozymerecognizes and binds a target RNA usually through complementarybase-pairing, and once bound to the correct site, acts enzymatically tocleave and inactivate the target RNA. Cleavage of a target RNA in such amanner will destroy its ability to direct synthesis of an encodedprotein if the cleavage occurs in the coding sequence. After a ribozymehas bound and cleaved its RNA target, it is typically released from thatRNA and so can bind and cleave new targets repeatedly.

6) Identifying Telomerase-Associated Proteins for Use as Modulators

In one embodiment of the invention, telomerase is used to identifytelomerase-associated proteins, i.e., telomerase accessory proteinswhich modulate or otherwise complement telomerase activity. As notedabove, these proteins or fragments thereof can modulate function bycausing the dissociation or preventing the association of the telomeraseenzyme complex, preventing the assembly of the telomerase complex,preventing hTRT from binding to its nucleic acid complement or to itsDNA template, preventing hTRT from binding nucleotides, or preventing,augmenting, or inhibiting any one, several or all of the partialactivities of the telomerase enzyme or hTRT protein, as described above.

One of skill in the art can use the methods of the invention to identifywhich portions (e.g., domains) of these telomerase-associating proteinscontact telomerase. In one embodiment of the invention, thesetelomerase-associating proteins or fragments thereof are used asmodulators of telomerase activity.

7) Telomerase-Associated Proteins as Dominant Negative Mutants

In one embodiment of the invention, telomerase-associated proteins areused as modulators of telomerase activity. Telomerase-associatedproteins include chromosomal structures, such as histones, nuclearmatrix proteins, cell division and cell cycle control proteins, and thelike. Other telomerase-associated proteins which can be used asmodulators for the purpose of the invention include the p80 and p95proteins and their human homologs, such as TP1 and TRF-1 (Chong, 1995,Science 270:1663-1667). In addition, fragments of thesetelomerase-associated proteins can be identified by the skilled artisanin accordance with the methods of the invention and used as modulatorsof telomerase activity.

8) Dominant Negative Mutants

Eight highly conserved motifs have been identified between TRTs ofdifferent non-human species, as described above (see also Lingner (1997)Science 276:561-567). FIG. 4 shows a schematic of the human TRT aminoacid sequence (from pGRN121) and RT motifs as compared to S. pombeTrt1p, Euplotes p123 and S. cerevisiae Est2p. The present inventionprovides recombinant and synthetic nucleic acids in which the codons forthe conserved amino acid residues in each, alone or in conjunction withone or more additional codons, of all eight of these motifs has been achanged to each of the other codons. A variety of the resulting codingsequences express a non-functional hTRT. See, for instance, Example 16.Thus, the present invention provides, for example, a wide variety of“mutated” telomerase enzymes and TRT proteins which have a partialactivity but not full activity of telomerase. For example, one suchtelomerase is able to bind telomeric structures, but not bindtelomerase-associated RNA (i.e., hTR). If expressed at high enoughlevels, such a telomerase mutant can deplete a necessary telomerasecomponent (e.g., hTR) and thereby function as an inhibitor of wild-typetelomerase activity. A mutated telomerase acting in this manner is anantagonist or a so-called “dominant-negative” mutant.

9) Antibodies

In general, the antibodies of the invention can be used to identify,purify, or inhibit any or all activity of telomerase enzyme and hTRTprotein. Antibodies can act as antagonists of telomerase activity in avariety of ways, for example, by preventing the telomerase complex ornucleotide from binding to its DNA substrates, by preventing thecomponents of telomerase from forming an active complex, by maintaininga functional (telomerase complex) quaternary structure or by binding toone of the enzyme's active sites or other sites that have allostericeffects on activity (the different partial activities of telomerase aredescribed in detail elsewhere in this specification).

D) Modulator Synthesis

It is contemplated that the telomerase modulators of the invention willbe made using methods well known in the pharmaceutical arts, includingcombinatorial methods and rational drug design techniques.

1) Combinatorial Chemistry Methodology

The creation and simultaneous screening of large libraries of syntheticmolecules can be carried out using well-known techniques incombinatorial chemistry, for example, see van Breemen (1997) Anal Chem69:2159-2164; Lam (1997) Anticancer Drug Des 12:145-167 (1997).

As noted above, combinatorial chemistry methodology can be used tocreate vast numbers of oligonucleotides (or other compounds) that can berapidly screened for specific oligonucleotides (or compounds) that haveappropriate binding affinities and specificities toward any target, suchas the TRT proteins of the invention, can be utilized (for generalbackground information Gold (1995) J. of Biol. Chem. 270:13581-13584).

2) Rational Drug Design

Rational drug design involves an integrated set of methodologies thatinclude structural analysis of target molecules, synthetic chemistries,and advanced computational tools. When used to design modulators, suchas antagonists/inhibitors of protein targets, such as telomerase enzymeand hTRT protein, the objective of rational drug design is to understanda molecule's three-dimensional shape and chemistry. Rational drug designis aided by X-ray crystallographic data or NMR data, which can now bedetermined for the hTRT protein and telomerase enzyme in accordance withthe methods and using the reagents provided by the invention.Calculations on electrostatics, hydrophobicities and solventaccessibility is also helpful. See, for example, Coldren (1997) Proc.Natl. Acad. Sci. USA 94:6635-6640.

E) Kits

The invention also provides kits that can be used to aid in determiningwhether a test compound is a modulator of a TRT activity. The kit willtypically include one or more of the following components: asubstantially purified TRT polypeptide or polynucleotide (includingprobes and primers); a plasmid capable of expressing a TRT (e.g., hTRT)when introduced into a cell or cell-free expression system; a plasmidcapable of expressing a TR (e.g., hTR) when introduced into a cell orcell-free expression system; cells or cell lines; a composition todetect a change in TRT activity; and, an instructional material teachinga means to detect and measure a change in the TRT activity, indicatingthat a change in the telomerase activity in the presence of the testcompound is an indicator that the test compound modulates the telomeraseactivity, and one or more containers. The kit can also include means,such as TRAP assay reagents or reagents for a quantitative polymerasechain reaction assay, to measure a change in TRT activity. The kit mayalso include instructional material teaching a means to detect andmeasure a change in the TRT activity, indicating that a change in thetelomerase activity in the presence of the test compound is an indicatorthat the test compound modulates the telomerase activity.

XI. Transgenic Organisms (Telomerase Knockout Cells and Animal Models)

The invention also provides transgenic non-human multicellular organisms(e.g., plants and non-human animals) or unicellular organisms (e.g.,yeast) comprising an exogenous TRT gene sequence, which may be a codingsequence or a regulatory (e.g., promoter) sequence. In one embodiment,the organism expresses an exogenous TRT polypeptide, having a sequenceof a human TRT protein. In a related embodiment, the organism alsoexpresses a telomerase RNA component (e.g., hTR).

The invention also provides unicellular and multicellular organisms (orcells therefrom) in which at least one gene encoding a telomerasecomponent (e.g., TRT or TR) or telomerase-associated protein is mutatedor deleted (i.e., in a coding or regulatory region) such that nativetelomerase is not expressed, or is expressed at reduced levels or withdifferent activities when compared to wild-type cells or organisms. Suchcells and organisms are often referred to as “gene knock-out” cells ororganisms.

The invention further provides cells and organisms in which anendogenous telomerase gene (e.g., murine TRT) is either present oroptionally mutated or deleted and an exogenous telomerase gene orvariant (e.g., human TRT) is introduced and expressed. Cells andorganisms of this type will be useful, for example, as model systems foridentifying modulators of hTRT activity or expression; determining theeffects of mutations in telomerase component genes, and other uses suchas determining the developmental timing and tissue location oftelomerase activity (e.g., for assessing when to administer a telomerasemodulator and for assessing any potential side effects).

Examples of multicellular organisms include plants, insects, andnonhuman animals such as mice, rats, rabbits, monkeys, apes, pigs, andother nonhuman mammals. An example of a unicellular organism is a yeast.

Methods for alteration or disruption of specific genes (e.g., endogenousTRT genes) are well known to those of skill, see, e.g., Baudin et al.,1993, Nucl. Acids Res. 21:3329; Wach et al., 1994, Yeast 10:1793;Rothstein, 1991, Methods Enzymol. 194:281; Anderson, 1995, Methods CellBiol. 48:31; Pettitt et al., 1996, Development 122:4149-4157;Ramirez-Solis et al., 1993, Methods Enzymol. 225:855; and Thomas et al.,1987, Cell 51:503, each of which is incorporated herein by reference inits entirety for all purposes.

The “knockout” cells and animals of the invention include cells andanimals in which one or several units of the endogenous telomeraseenzyme complex have been deleted or inhibited. Reconstitution oftelomerase activity will save the cell or animal from senescence or, forcancer cells, cell death caused by its inability to maintain telomeres.Methods of altering the expression of endogenous genes are well known tothose of skill in the art. Typically, such methods involve altering orreplacing all or a portion of the regulatory sequences controllingexpression of the particular gene to be regulated. The regulatorysequences, e.g., the native promoter can be altered. The conventionaltechnique for targeted mutation of genes involves placing a genomic DNAfragment containing the gene of interest into a vector, followed bycloning of the two genomic arms associated with the targeted gene arounda selectable neomycin-resistance cassette in a vector containingthymidine kinase. This “knock-out” construct is then transfected intothe appropriate host cell, i.e., a mouse embryonic stem (ES) cell, whichis subsequently subjected to positive selection (using G418, forexample, to select for neomycin-resistance) and negative selection(using, for example, FIAU to exclude cells lacking thymidine kinase),allowing the selection of cells which have undergone homologousrecombination with the knockout vector. This approach leads toinactivation of the gene of interest. See, e.g., U.S. Pat. Nos.5,464,764; 5,631,153; 5,487,992; and, 5,627,059.

“Knocking out” expression of an endogenous gene can also be accomplishedby the use of homologous recombination to introduce a heterologousnucleic acid into the regulatory sequences (e.g., promoter) of the geneof interest. To prevent expression of functional enzyme or product,simple mutations that either alter the reading frame or disrupt thepromoter can be suitable. To up-regulate expression, a native promotercan be substituted with a heterologous promoter that induces higherlevels of transcription. Also, “gene trap insertion” can be used todisrupt a host gene, and mouse ES cells can be used to produce knockouttransgenic animals, as described for example, in Holzschu (1997)Transgenic Res 6: 97-106.

Altering the expression of endogenous genes by homologous recombinationcan also be accomplished by using nucleic acid sequences comprising thestructural gene in question. Upstream sequences are utilized fortargeting heterologous recombination constructs. Utilizing TRTstructural gene sequence information, such as SEQ ID NO:1, one of skillin the art can create homologous recombination constructs with onlyroutine experimentation. Homologous recombination to alter expression ofendogenous genes is described in U.S. Pat. No. 5,272,071, and WO91/09955, WO 93/09222, WO 96/29411, WO 95/31560, and WO 91/12650.Homologous recombination in mycobacteria is described by Azad (1996)Proc. Natl. Acad. Sci. USA 93:4787; Baulard (1996) J.Bacteriol.178:3091; and Pelicic (1996) Mol. Microbiol. 20:919.Homologous recombination in animals has been described by Moynahan(1996) Hum. Mol. Genet. 5:875, and in plants by Offringa (1990) EMBO J.9:3077.

XII. GLOSSARY

The following terms are defined infra to provide additional guidance toone of skill in the practice of the invention: adjuvant, allele (&allelic sequence), amino acids (including hydrophobic, polar, charged),conservative substitution, control elements (& regulatory sequences),derivatized, detectable label, elevated level, epitope, favorable andunfavorable prognosis, fusion protein, gene product, hTR, immortal,immunogen and immunogenic, isolated, modulator, motif, nucleic acid (&polynucleotide), oligonucleotides (& oligomers), operably linked,polypeptide, probe (including nucleic acid probes & antibody probes),recombinant, selection system, sequence, specific binding, stringenthybridization conditions (& stringency), substantial identity (&substantial similarity), substantially pure (& substantially purified),telomerase-negative and telomerase-positive cells, telomerase catalyticactivity, telomerase-related, and test compound.

As used herein, the term “adjuvant” refers to its ordinary meaning ofany substance that enhances the immune response to an antigen with whichit is mixed. Adjuvants useful in the present invention include, but arenot limited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium parvumare potentially useful adjuvants.

As used herein, the terms “allele” or “allelic sequence” refer to analternative form of a nucleic acid sequence (i.e., a nucleic acidencoding hTRT protein). Alleles result from mutations (i.e., changes inthe nucleic acid sequence), and generally produce altered and/ordifferently regulated mRNAs or polypeptides whose structure and/orfunction may or may not be altered. Common mutational changes that giverise to alleles are generally ascribed to natural deletions, additions,or substitutions of nucleotides that may or may not affect the encodedamino acids. Each of these types of changes may occur alone, incombination with the others, or one or more times within a given gene,chromosome or other cellular nucleic acid. Any given gene may have no,one or many allelic forms. As used herein, the term “allele” refers toeither or both a gene or an mRNA transcribed from the gene.

As used herein, “amino acids” are sometimes specified using the standardone letter code: Alanine (A), Serine (S), Threonine (T), Aspartic acid(D), Glutamic acid (E) Asparagine (N), Glutamine (Q), Arginine (R),Lysine (K), Isoleucine (I), Leucine (L), Methionine (M), Valine (V),Phenylalanine (F), Tyrosine (Y), Tryptophan (W), Proline (P), Glycine(G), Histidine (H), Cysteine (C). Synthetic and non-naturally occurringamino acid analogues (and/or peptide linkages) are included.

As used herein, “Hydrophobic amino acids” refers to A, L, I, V, P, F, W,and M. As used herein, “polar amino acids” refers to G, S, T, Y, C, N,and Q. As used herein, “charged amino acids” refers to D, E, H, K, andR.

As used herein, “conservative substitution”, when describing a proteinrefers to a change in the amino acid composition of the protein thatdoes not substantially alter the protein's activity. Thus,“conservatively modified variations” of a particular amino acid sequencerefers to amino acid substitutions of those amino acids that are notcritical for protein activity or substitution of amino acids with otheramino acids having similar properties (e.g., acidic, basic, positivelyor negatively charged, polar or non-polar, etc.) such that thesubstitutions of even critical amino acids does not substantially alteractivity. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following six groupseach contain amino acids that are conservative substitutions for oneanother: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid(D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine(V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W) (see also,Creighton (1984) Proteins, W.H. Freeman and Company). One of skill inthe art will appreciate that the above-identified substitutions are notthe only possible conservative substitutions. For example, one mayregard all charged amino acids as conservative substitutions for eachother whether they are positive or negative. In addition, individualsubstitutions, deletions or additions which alter, add or delete asingle amino/acid or a small percentage of amino acids in an encodedsequence can also be “conservatively modified variations”. One can alsomake a “conservative substitution” in a recombinant protein by utilizingone or more codons that differ from the codons employed by the native orwild-type gene. In this instance, a conservative substitution alsoincludes substituting a codon for an amino acid with a different codonfor the same amino acid.

As used herein, “control elements” or “regulatory sequences” includeenhancers, promoters, transcription terminators, origins of replication,chromosomal integration sequences, 5′ and 3′ untranslated regions, withwhich proteins or other biomolecules interact to carry out transcriptionand translation. For eukaryotic cells, the control sequences willinclude a promoter and preferably an enhancer, e.g., derived fromimmunoglobulin genes, SV40, cytomegalovirus, and a polyadenylationsequence, and may include splice donor and acceptor sequences. Dependingon the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used.

As used herein, a “derivatized” polynucleotide, oligonucleotide, ornucleic acid refers to oligo- and polynucleotides that comprise aderivatized substituent. In some embodiments, the substituent issubstantially non-interfering with respect to hybridization tocomplementary polynucleotides. Derivatized oligo- or polynucleotidesthat have been modified with appended chemical substituents (e.g., bymodification of an already synthesized oligo- or poly-nucleotide, or byincorporation of a modified base or backbone analog during synthesis)may be introduced into a metabolically active eukaryotic cell tohybridize with an hTRT DNA, RNA, or protein where they produce analteration or chemical modification to a local DNA, RNA, or protein.Alternatively, the derivatized oligo or polynucleotides may interactwith and alter hTRT polypeptides, telomerase-associated proteins, orother factors that interact with hTRT DNA or hTRT gene products, oralter or modulate expression or function of hTRT DNA, RNA or protein.Illustrative attached chemical substituents include: europium (m)texaphyrin, cross-linking agents, psoralen, metal chelates (e.g.,iron/EDTA chelate for iron catalyzed cleavage), topoisomerases,endonucleases, exonucleases, ligases, phosphodiesterases, photodynamicporphyrins, chemotherapeutic drugs (e.g., adriamycin, doxirubicin),intercalating agents, base-modification agents, immunoglobulin chains,and oligonucleotides. Iron/EDTA chelates are chemical substituents oftenused where local cleavage of a polynucleotide sequence is desired(Hertzberg et al., 1982, J. Am. Chem. Soc. 104: 313; Hertzberg andDervan, 1984, Biochemistry 23: 3934; Taylor et al., 1984, Tetrahedron40: 457; Dervan, 1986, Science 232: 464. Illustrative attachmentchemistries include: direct linkage, e.g., via an appended reactiveamino group (Corey and Schultz (1988) Science 238: 1401, which isincorporated herein by reference) and other direct linkage chemistries,although streptavidin/biotin and digoxigenin/anti-digoxigenin antibodylinkage methods can also be used. Methods for linking chemicalsubstituents are provided in U.S. Pat. Nos. 5,135,720, 5,093,245, and5,055,556, which are incorporated herein by reference. Other linkagechemistries may be used at the discretion of the practitioner.

As used herein, a “detectable label” has the ordinary meaning in the artand refers to an atom (e.g., radionuclide), molecule (e.g.,fluorescein), or complex, that is or can be used to detect (e.g., due toa physical or chemical property), indicate the presence of a molecule orto enable binding of another molecule to which it is covalently bound orotherwise associated. The term “label” also refers to covalently boundor otherwise associated molecules (e.g., a biomolecule such as anenzyme) that act on a substrate to produce a detectable atom, moleculeor complex. Detectable labels suitable for use in the present inventioninclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Labels useful in the present invention include biotin for staining withlabeled streptavidin conjugate, magnetic beads (e.g., Dynabeads™),fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, greenfluorescent protein, enhanced green fluorescent protein, lissamine,phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX [Amersham], SyBRGreen I & II [Molecular Probes], and the like), radiolabels (e.g.,³H,¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., hydrolases, particularlyphosphatases such as alkaline phosphatase, esterases and glycosidases,or oxidoreductases, particularly peroxidases such as horse radishperoxidase, and others commonly used in ELISAs), substrates, cofactors,inhibitors, chemiluminescent groups, chromogenic agents, andcolorimetric labels such as colloidal gold or colored glass or plastic(e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Means ofdetecting such labels are well known to those of skill in the art. Thus,for example, radiolabels and chemiluminescent labels may be detectedusing photographic film or scintillation counters, fluorescent markersmay be detected using a photodetector to detect emitted light (e.g., asin fluorescence-activated cell sorting). Enzymatic labels are typicallydetected by providing the enzyme with a substrate and detecting thereaction product produced by the action of the enzyme on the substrate,and colorimetric labels are detected by simply visualizing the coloredlabel. Thus, a label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. The label may be coupled directly or indirectly to thedesired component of the assay according to methods well known in theart. Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to an anti-ligand (e.g., streptavidin)molecule which is either inherently detectable or covalently bound to asignal generating system, such as a detectable enzyme, a fluorescentcompound, or a chemiluminescent compound. A number of ligands andanti-ligands can be used. Where a ligand has a natural anti-ligand, forexample, biotin, thyroxine, and cortisol, it can be used in conjunctionwith the labeled, naturally occurring anti-ligands. Alternatively, anyhaptenic or antigenic compound can be used in combination with anantibody. The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Means of detecting labels are well known to those of skillin the art. Thus, for example, where the label is a radioactive label,means for detection include a scintillation counter, photographic filmas in autoradiography, or storage phosphor imaging. Where the label is afluorescent label, it may be detected by exciting the fluorochrome withthe appropriate wavelength of light and detecting the resultingfluorescence. The fluorescence may be detected visually, by means ofphotographic film, by the use of electronic detectors such as chargecoupled devices (CCDs) or photomultipliers and the like. Similarly,enzymatic labels may be detected by providing the appropriate substratesfor the enzyme and detecting the resulting reaction product. Also,simple colorimetric labels may be detected by observing the colorassociated with the label. It will be appreciated that when pairs offluorophores are used in an assay, it is often preferred that the theyhave distinct emission patterns (wavelengths) so that they can be easilydistinguished.

The phrase “elevated level” refers to an amount of hTRT gene product (orother specified substance or activity) in a cell that is elevated orhigher than the level in a reference standard, e.g., for diagnosis, thelevel in normal, telomerase-negative cells in an individual or in otherindividuals not suffering from the condition, and for prognosis, thelevel in tumor cells from a variety of grades or classes of, e.g.,tumors.

As used herein, the term “epitope” has its ordinary meaning of a site onan antigen recognized by an antibody. Epitopes are typically segments ofamino acids which are a small portion of the whole protein. Epitopes maybe conformational (i.e., discontinuous). That is, they may be formedfrom amino acids encoded by noncontiguous parts of a primary sequencethat have been juxtaposed by protein folding.

The terms “favorable prognosis” and “unfavorable prognosis” are known inthe art. In the context of cancers, “favorable prognosis” means thatthere is a likelihood of tumor regression or longer survival times forpatients with a favorable prognosis relative to those with unfavorableprognosis, whereas “unfavorable prognosis” means that the tumor islikely to be more aggressive, i.e., grow faster and/or metastasize,resulting in a poor outcome or a more rapid course of diseaseprogression for the patient.

As used herein, the term “fusion protein,” refers to a compositeprotein, i.e., a single contiguous amino acid sequence, made up of two(or more) distinct, heterologous polypeptides which are not normallyfused together in a single amino acid sequence. Thus, a fusion proteinmay include a single amino acid sequence that contains two entirelydistinct amino acid sequences or two similar or identical polypeptidesequences, provided that these sequences are not normally found togetherin the same configuration in a single amino acid sequence found innature. Fusion proteins may generally be prepared using eitherrecombinant nucleic acid methods, i.e., as a result of transcription andtranslation of a recombinant gene fusion product, which fusion comprisesa segment encoding a polypeptide of the invention and a segment encodinga heterologous protein, or by chemical synthesis methods well known inthe art. The non-hTRT region(s) of the fusion protein can be fused tothe amino terminus of the hTRT polypeptide or the carboxyl terminus, orboth or the non-hTRT region can be inserted into the interior of theprotein sequence (by moiety inserting or by replacing amino acids) orcombinations of the foregoing can be performed.

As used herein, the term “gene product” refers to an RNA moleculetranscribed from a gene, or a protein encoded by the gene or translatedfrom the RNA.

As used herein, “hTR” (human telomerase RNA) refers to the RNA componentof human telomerase and any naturally occurring alleles and variants orrecombinant variants. hTR is described in detail in U.S. Pat. No.5,583,016 which is incorporated herein by reference in its entirety andfor all purposes.

As used herein, the term “immortal,” when referring to a cell, has itsnormal meaning in the telomerase art and refers to cells that haveapparently unlimited replicative potential. Immortal can also refer tocells with increased proliferative capacity relative to their unmodifiedcounterparts. Examples of immortal human cells are malignant tumorcells, germ line cells, and certain transformed human cell linescultured in vitro (e.g., cells that have become immortal followingtransformation by viral oncogenes or otherwise). In contrast, mostnormal human somatic cells are mortal, i.e., have limited replicativepotential and become senescent after a finite number of cell divisions.

As used herein, the terms “immunogen” and “immunogenic” have theirordinary meaning in the art, i.e, an immunogen is a molecule, such as aprotein or other antigen, that can elicit an adaptive immune responseupon injection into a person or an animal.

As used herein, “isolated,” when referring to a molecule or composition,such as, for example, an RNP (e.g., at least one protein and at leastone RNA), means that the molecule or composition is separated from atleast one other compound, such as a protein, other RNAs, or othercontaminants with which it is associated in vivo or in its naturallyoccurring state. Thus, an RNP is considered isolated when the RNP hasbeen isolated from any other component with which it is naturallyassociated, e.g., cell membrane, as in a cell extract. An isolatedcomposition can, however, also be substantially pure.

As used herein, “modulator” refers to any synthetic or natural compoundor composition that can change in any way either or both the “full” orany “partial activity” of a telomerase reverse transcriptase (TRT). Amodulator can be an agonist or an antagonist. A modulator can be anyorganic and inorganic compound; including, but not limited to, forexample, small molecules, peptides, proteins, sugars, nucleic acids,fatty acids and the like.

As used herein, “motif” refers to a sequence of contiguous amino acids(or to a nucleic acid sequence that encodes a sequence of contiguousamino acids) that defines a feature or structure in a protein that iscommon to or conserved in all proteins of a defined class or type. Themotif or consensus sequence may include both conserved and non-conservedresidues. The conserved residues in the motif sequence indicate that theconserved residue or class (i.e., hydrophobic, polar, non-polar, orother class) of residues is typically present at the indicated locationin each protein (or gene or mRNA) of the class of proteins defined bythe motif. Motifs can differ in accordance with the class of proteins.Thus, for example, the reverse transcriptase enzymes form a class ofproteins than can be defined by one or more motifs, and this classincludes telomerase enzymes. However, the telomerase enzymes can also bedefined as the class of enzymes with motifs characteristic for thatclass. Those of skill recognize that the identification of a residue asa conserved residue in a motif does not mean that every member of theclass defined by the motif has the indicated residue (or class ofresidues) at the indicated position, and that one or more members of theclass may have a different residue at the conserved position.

As used herein, the terms “nucleic acid” and “polynucleotide” are usedinterchangeably. Use of the term “polynucleotide” is not intended toexclude oligonucleotides (i.e., short polynucleotides) and can alsorefer to synthetic and/or non-naturally occurring nucleic acids (i.e.,comprising nucleic acid analogues or modified backbone residues orlinkages).

As used herein “oligonucleotides” or “oligomers” refer to a nucleic acidsequence of approximately 7 nucleotides or greater, and as many asapproximately 100 nucleotides, which can be used as a primer, probe oramplimer. Oligonucleotides are often between about 10 and about 50nucleotides in length, more often between about 14 and about 35nucleotides, very often between about 15 and about 25 nucleotides, andthe terms oligonucleotides or oligomers can also refer to syntheticand/or non-naturally occurring nucleic acids (i.e., comprising nucleicacid analogues or modified backbone residues or linkages).

As used herein, the term “operably linked,” refers to a functionalrelationship between two or more nucleic acid (e.g., DNA) segments: forexample, a promoter or enhancer is operably linked to a coding sequenceif it stimulates the transcription of the sequence in an appropriatehost cell or other expression system. Generally, sequences that areoperably linked are contiguous, and in the case of a signal sequenceboth contiguous and in reading phase. However, enhancers need not belocated in close proximity to the coding sequences whose transcriptionthey enhance.

As used herein, the term “polypeptide” is used interchangeably hereinwith the term “protein,” and refers to a polymer composed of amino acidresidues linked by amide linkages, including synthetic,naturally-occurring and non-naturally occurring analogs thereof (aminoacids and linkages). Peptides are examples of polypeptides.

As used herein, a “probe” refers to a molecule that specifically bindsanother molecule. One example of a probe is a “nucleic acid probe” thatspecifically binds (i.e., anneals or hybridizes) to a substantiallycomplementary nucleic acid. Another example of a probe is an “antibodyprobe” that specifically binds to a corresponding antigen or epitope.

As used herein, “recombinant” refers to a polynucleotide synthesized orotherwise manipulated in vitro (e.g., “recombinant polynucleotide”), tomethods of using recombinant polynucleotides to produce gene products incells or other biological systems, or to a polypeptide (“recombinantprotein”) encoded by a recombinant polynucleotide.

As used herein, a “selection system,” in the context of stablytransformed cell lines, refers to a method for identifying and/orselecting cells containing a recombinant nucleic acid of interest. Alarge variety of selection systems are known for identification oftransformed cells and are suitable for use with the present invention.For example, cells transformed by plasmids or other vectors can beselected by resistance to antibiotics conferred by genes contained onthe plasmids, such as the well known amp, gpt, neo and hyg genes, orother genes such as the herpes simplex virus thymidine kinase (Wigler etal., Cell 11:223-32 [1977]) and adenine phosphoribosyltransferase (Lowyet al., Cell 22:817 [1980]) genes which can be employed in tk− or aprt−cells, respectively. Also, antimetabolite, antibiotic or herbicideresistance can be used as the basis for selection; for example, dhfrwhich confers resistance to methotrexate and is also useful for geneamplification (Wigler et al., Proc. Natl. Acad. Sci., 77:3567 [1980]);npt, which confers resistance to the aminoglycosides neomycin and G-418(Colbere-Garapin et al., J. Mol. Biol., 150:1 [1981]) and als or pat,which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, in McGraw Hill Yearbook ofScience and Technology, McGraw Hill, New York N.Y., pp 191-196, [1992]).Additional selectable genes have been described, for example, hygromycinresistance-conferring genes, trpB, which allows cells to utilize indolein place of tryptophan, or hisD, which allows cells to utilize histinolin place of histidine (Hartman and Mulligan, Proc. Natl. Acad. Sci.,85:8047 [1988]). Recently, the use of visible markers has gainedpopularity with such markers as anthocyanins, beta-glucuronidase and itssubstrate, GUS, and luciferase and its substrate, luciferin, beingwidely used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system (Rhodes et al., Meth. Mol. Biol., 55:121 [1995]).

As used herein, the “sequence” of a gene (unless specifically statedotherwise), nucleic acid, protein, or peptide refers to the order ofnucleotides in either or both strands of a double-stranded DNA molecule,e.g., the sequence of both the coding strand and its complement, or of asingle-stranded nucleic acid molecule, or to the order of amino acids ina peptide or protein.

As used herein, “specific binding” refers to the ability of onemolecule, typically an antibody or polynucleotide, to contact andassociate with another specific molecule even in the presence of manyother diverse molecules. For example, a single-stranded polynucleotidecan specifically bind to a single-stranded polynucleotide that iscomplementary in sequence, and an antibody specifically binds to (or “isspecifically immunoreactive with”) its corresponding antigen.

As used herein, “stringent hybridization conditions” or “stringency”refers to conditions in a range from about 5° C. to about 20° C. or 25°C. below the melting temperature (T_(m)) of the target sequence and aprobe with exact or nearly exact complementarity to the target. As usedherein, the melting temperature is the temperature at which a populationof double-stranded nucleic acid molecules becomes half-dissociated intosingle strands. Methods for calculating the T_(m) of nucleic acids arewell known in the art (see, e.g., Berger and Kimmel (1987) Methods inEnzymology, Vol. 152: Guide to Molecular Cloning Techniques, San Diego:Academic Press, Inc. and Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual, 2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratoryhereinafter, “Sambrook”), both incorporated herein by reference). Asindicated by standard references, a simple estimate of the T_(m) valuemay be calculated by the equation: T_(m)=81.5+0.41(% G+C), when anucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson andYoung, Quantitative Filter Hybridization in Nucleic Acid Hybridization(1985)). Other references include more sophisticated computations whichtake structural as well as sequence characteristics into account for thecalculation of T_(m). The melting temperature of a hybrid (and thus theconditions for stringent hybridization) is affected by various factorssuch as the length and nature (DNA, RNA, base composition) of the probeand nature of the target (DNA, RNA, base composition, present insolution or immobilized, and the like), and the concentration of saltsand other components (e.g., the presence or absence of formamide,dextran sulfate, polyethylene glycol). The effects of these factors arewell known and are discussed in standard references in the art, e.g.,Sambrook, supra and Ausubel et al. supra. Typically, stringenthybridization conditions are salt concentrations less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion at pH 7.0 to 8.3,and temperatures at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). As noted, stringent conditions may also beachieved with the addition of destabilizing agents such as formamide, inwhich case lower temperatures may be employed.

As used herein, the term “substantial identity,” “substantial sequenceidentity,” or “substantial similarity” in the context of nucleic acids,refers to a measure of sequence similarity between two polynucleotides.Substantial sequence identity can be determined by hybridization understringent conditions, by direct comparison, or other means. For example,two polynucleotides can be identified as having substantial sequenceidentity if they are capable of specifically hybridizing to each otherunder stringent hybridization conditions. Other degrees of sequenceidentity (e.g., less than “substantial”) can be characterized byhybridization under different conditions of stringency. Alternatively,substantial sequence identity can be described as a percentage identitybetween two nucleotide (or polypeptide) sequences. Two sequences areconsidered substantially identical when they are at least about 60%identical, preferably at least about 70% identical, or at least about80% identical, or at least about 90% identical, or at least about 95% or98% to 100% identical. Percentage sequence (nucleotide or amino acid)identity is typically calculated by determining the optimal alignmentbetween two sequences and comparing the two sequences. For example anexogenous transcript used for protein expression can be described ashaving a certain percentage of identity or similarity compared to areference sequence (e.g., the corresponding endogenous sequence).Optimal alignment of sequences may be conducted using the local homologyalgorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by thehomology alignment algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48: 443, by the search for similarity method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. U.S.A. 85: 2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by inspection. The best alignment (i.e.,resulting in the highest percentage of identity) generated by thevarious methods is selected. Typically these algorithms compare the twosequences over a “comparison window” (usually at least 18 nucleotides inlength) to identify and compare local regions of sequence similarity,thus allowing for small additions or deletions (i.e., gaps). Additionsand deletions are typically 20 percent or less of the length of thesequence relative to the reference sequence, which does not compriseadditions or deletions. It is sometimes desirable to describe sequenceidentity between two sequences in reference to a particular length orregion (e.g., two sequences may be described as having at least 95%identity over a length of at least 500 basepairs). Usually the lengthwill be at least about 50, 100, 200, 300, 400 or 500 basepairs, aminoacids, or other residues. The percentage of sequence identity iscalculated by comparing two optimally aligned sequences over the regionof comparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T, C, G, or U) occurs in bothsequences to yield the number of matched positions, and determining thenumber (or percentage) of matched positions as compared to the totalnumber of bases in the reference sequence or region of comparison. Anadditional algorithm that is suitable for determining sequencesimilarity is the BLAST algorithm, which is described in Altschul (1990)J. Mol. Biol. 215: 403-410; and Shpaer (1996) Genomics 38:179-191.Software for performing BLAST analyses is publicly available at theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence that either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al, supra.). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a wordlength (W) of11, the BLOSUM62 scoring matrix (see Henikoff (1992) Proc. Natl. Acad.Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10,M=5, N=−4, and a comparison of both strands. The term BLAST refers tothe BLAST algorithm which performs a statistical analysis of thesimilarity between two sequences; see, e.g., Karlin (1993) Proc. Natl.Acad. Sci. USA 90:5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid can be considered similar to a TRT nucleic acid if the smallest sumprobability in a comparison of the test nucleic acid to an TRT nucleicacid is less than about 0.5, 0.2, 0.1, 0.01, or 0.001. Alternatively,another indication that two nucleic acid sequences are similar is thatthe polypeptide which the first nucleic acid encodes is immunologicallycross reactive with the polypeptide encoded by the second nucleic acid.It will be recognized that homologous non-human TRT polynucleotides mayhave less that “substantial” nucleotide identity in certain regions, asthe term “substantial identity” is defined herein. For example, EuplotesTRT is substantially less than about 60% identical to the hTRTpolynucleotide of SEQ ID NO:1 in certain regions, although the two genesare homologs.

As used herein, the terms “substantial identity,” “substantial sequenceidentity,” or “substantial similarity” in the context of a polypeptide,refers to a degree of similarity between two polypeptides in which apolypeptides comprises a sequence with at least 70% sequence identity toa reference sequence, or 80%, or 85% or up to 100% sequence identity tothe reference sequence, or most preferably 90% identity over acomparison window of about 10-20 amino acid residues. Amino acidsequence similarity, or sequence identity, is determined by optimizingresidue matches, if necessary, by introducing gaps as required. SeeNeedleham et al. (1970) J. Mol. Biol. 48: 443-453; and Sankoff et al.,1983, Time Warps, String Edits, and Macromolecules, The Theory andPractice of Sequence Comparison, Chapter One, Addison-Wesley, Reading,Mass.; and software packages from IntelliGenetics, Mountain View,Calif., and the University of Wisconsin Genetics Computer Group,Madison, Wis. As will be apparent to one of skill, the terms“substantial identity”, “substantial similarity” and “substantialsequence identity” can be used interchangeably with regard topolypeptides or polynucleotides. It will be recognized that homologousnon-human TRT polypeptides may have less that “substantial” sequenceidentity in certain regions, as the term “substantial identity” isdefined herein. For example, Euplotes TRT protein is substantially lessthan about 60% identical to the hTRT polynucleotide of SEQ ID NO:2 incertain regions, although the two genes are homologs. In the context ofTRT polypeptides from different species, for example, “significanthomology” at the amino acid sequence means at least about 20% sequenceidentity in region of about 20 to about 40 residues, or at least about40% sequence identity in region of at least about 20% sequence identity.

As used herein, the term “substantially pure,” or “substantiallypurified,” when referring to a composition comprising a specifiedreagent, such as an antibody (e.g. an anti-hTRT antibody), means thatthe specified reagent is at least about 75%, or at least about 90%, orat least about 95%, or at least about 99% or more of the composition(not including, e.g., solvent or buffer). Thus, for example, a preferredimmunoglobulin preparation of the invention that specifically binds anhTRT polypeptide is substantially purified.

As used herein, a “telomerase negative” cell is one in which telomeraseis not expressed, i.e., no telomerase catalytic activity can be detectedusing a conventional assay or a TRAP assay for telomerase catalyticactivity. As used herein, a “telomerase positive” cell is a cell inwhich telomerase is expressed (i.e. telomerase activity can bedetected).

As used herein, a “telomerase-related” disease or condition is a diseaseor condition in a subject that is correlated with an abnormally highlevel of telomerase activity in cells of the individual, which caninclude any telomerase activity at all for most normal somatic cells, orwhich is correlated with a low level of telomerase activity that resultsin impairment of a normal cell function. Examples of telomerase-relatedconditions include, e.g., cancer (high telomerase activity in malignantcells) and infertility (low telomerase activity in germ-line cells).

As used herein, “test compound” or “agent” refers to any synthetic ornatural compound or composition. The term includes all organic andinorganic compounds; including, for example, small molecules, peptides,proteins, sugars, nucleic acids, fatty acids and the like.

XIII. EXAMPLES

The following examples are provided to illustrate the present invention,and not by way of limitation.

In the following sections, the following abbreviations apply: eq(equivalents); M (Molar); μM (micromolar); N (Normal); mol (moles); mmol(millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg(milligrams); μg (micrograms); ng (nanograms); l or L (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); ° C. (degrees Centigrade); RPN(ribonucleoprotein); mreN (2′-O-methylribonucleotides); dNTP(deoxyribonucleotide); dH₂O (distilled water); DDT (dithiothreitol);PMSF (phenylmethylsulfonyl fluoride); TE (10 mM Tris HCl, 1 mM EDTA,approximately pH 7.2); KGlu (potassium glutamate); SSC (salt and sodiumcitrate buffer); SDS (sodium dodecyl sulfate); PAGE (polyacrylamide gelelectrophoresis); Novex (Novex, San Diego, Calif.); BioRad (Bio-RadLaboratories, Hercules, Calif.); Pharmacia (Pharmacia Biotech,Piscataway, N.J.); Boehringer-Mannheim (Boehringer-Mannheim Corp.,Concord, Calif.); Amersham (Amersham, Inc., Chicago, Ill.); Stratagene(Stratagene Cloning Systems, La Jolla, Calif.); NEB (New EnglandBiolabs, Beverly, Mass.); Pierce (Pierce Chemical Co., Rockford, Ill.);Beckman (Beckman Instruments, Fullerton, Calif.); Lab Industries (LabIndustries, Inc., Berkeley, Calif.); Eppendorf (Eppendorf Scientific,Madison, Wis.); and Molecular Dynamics (Molecular Dynamics, Sunnyvale,Calif.).

Example 1 Isolation of Telomerase Proteins and Clones

The following example details the isolation of telomerase proteins andclones from various organisms, including the euplotes p. 123, hTRT, TRTand S. pombe TRT telomerase cDNA clones.

A. Background

i) Introduction

This section provides an overview of the purification and cloning of TRTgenes, which is described in greater detail in subsequent sections ofthis Example. While telomerase RNA subunits have been identified inciliates, yeast and mammals, protein subunits of the enzyme have notbeen identified as such prior to the present invention. Purification oftelomerase from the ciliated protozoan Euplotes aediculatus yielded twoproteins, termed p123 and p43 (see infra; Lingner (1996) Proc. Natl.Acad. Sci. U.S.A. 93:10712). Euplotes aediculatus is a hypotrichousciliate having a macronucleus containing about 8×10⁷ telomeres and about3×10⁵ molecules of telomerase. After purification, the active telomerasecomplex had a molecular mass of about 230 kD, corresponding to a 66 kDRNA subunit and two proteins of about 123 kD and 43 kD (Lingner (1996)supra). Photocross-linking experiments indicated that the larger p123protein was involved in specific binding of the telomeric DNA substrate(Lingner, (1996) supra).

The p123 and p43 proteins were sequenced and the cDNA clones whichencoded these proteins were isolated. These Euplotes sequences werefound to be unrelated to the Tetrahymena telomerase-associated proteinsp80 and p95. Sequence analysis of the Euplotes p123 revealed reversetranscriptase (RT) motifs. Furthermore, sequence analysis of theEuplotes p123 by comparison to other sequences revealed a yeast homolog,termed Est2 protein (Lingner (1997) Science 276:561). Yeast Est2 hadpreviously been shown to be essential for telomere maintenance in vivo(Lendvay (1996) Genetics 144:1399) but had not been identified as atelomerase catalytic protein. Site-specific mutagenesis demonstratedthat the RT motifs of yeast Est2 are essential for telomeric DNAsynthesis in vivo and in vitro (Lingner (1997) supra).

ii) Identifying and Characterizing S. pombe Telomerase

PCR amplification of S. pombe DNA was carried out with degeneratesequence primers designed from the Euplotes p123 RT motifs as describedbelow. Of the four prominent PCR products generated, a 120 base pairband encoded a peptide sequence homologous to p123 and Est2. This PCRproduct was used as a probe in colony hybridization and identified twooverlapping clones from an S. pombe genomic library and three from an S.pombe cDNA library. Sequence analysis revealed that none of the three S.pombe cDNA clones was full length, so RT-PCR was used to obtain thesequences encoding the protein's N-terminus.

Complete sequencing of these clones revealed a putative S. pombetelomerase RT gene, trt1. The complete nucleotide sequence of trt1 hasbeen deposited in GenBank, accession number AF015783 (see FIG. 15).

To test S. pombe trt1 (as a catalytic subunit, two deletion constructswere created. Analysis of the sequence showed that trt1 encoded a basicprotein with a predicted molecular mass of 116 kD. It was found thathomology with p123 and Est2 was especially high in the seven reversetranscriptase motifs, underlined and designated as motifs 1, 2, A, B, C,D, and E (see FIG. 63). An additional telomerase-specific motif,designated the T-motif, was also found. Fifteen introns, ranging in sizefrom 36 to 71 base pairs, interrupted the coding sequence.

To test S. pombe trt1 as a catalytic subunit, two deletion constructswere created. One removed only motifs B through D in the RT domains. Thesecond removed 99% of the open reading frame.

Haploid cells grown from S. pombe spores of both mutants showedprogressive telomere shortening to the point where hybridization totelomeric repeats became almost undetectable. A trt1⁺/trt1⁻ diploid wassporulated and the resulting tetrads were dissected and germinated on ayeast extract medium supplemented with amino acids (a YES plate, Alfa(1993) Experiments with Fission Yeast, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). Colonies derived from each spore weregrown at 32° C. for three days, and streaked successively to fresh YESplates every three days. A colony from each round was placed in six mlof YES liquid culture at 32° C. and grown to stationary phase. GenomicDNA was prepared. After digestion with ApaI, DNA was subjected toelectrophoresis on a 2.3% agarose gel, stained with ethidium bromide toconfirm approximately equal loading in each lane, then transferred to anylon membrane and hybridized to a telomeric DNA probe.

Senescence was indicated by the delayed onset of growth or failure togrow on agar (typically at the fourth streak-out after germination) andby colonies with increasingly ragged edges (colony morphology shown inFIG. 22C) and by increasingly high fractions of elongated cells (asshown in FIG. 22D). Cells were plated on Minimal Medium (Alfa (1993)supra) with glutamic acid substituted for ammonium chloride for two daysat 32° C. prior to photography.

When individual enlarged cells were separated on the dissectingmicroscope, the majority were found to undergo no further division. Thesame telomerase negative (trt1⁻) cell population always containednormal-sized cells which continued to divide, but which frequentlyproduced non-dividing progeny. The telomerase-negative survivors may usea recombinational mode of telomere maintenance as documented in buddingyeast strains that have various telomere-replication genes deleted(Lendvay (1996) supra, Lundblad (1993) Cell 73:347).

iii) Identifying and Characterizing Human Telomerase

An EST (expressed sequence tag) derived from human telomerase reversetranscriptase (hTRT) cDNA was identified by a BLAST search of the dbEST(expressed sequence tag) Genbank database using the Euplotes 123 kDapeptide and nucleic acid sequences, as well as the Schizosaccharomycesprotein and corresponding cDNA (tez1) sequences. The EST, designatedGenbank AA28196, is 389 nucleotides long and it corresponds to positions1679 to 2076 of clone 712562 (FIG. 18), was obtained from the I.M.A.G.E.Consortium (Human Genome Center, DOE, Lawrence Livermore NationalLaboratory, Livermore, Calif.). This clone was obtained from a cDNAlibrary of germinal B cells derived by flow sorting of tonsil cells.Complete sequencing of this hTRT cDNA clone showed all eight telomeraseRT (TRT) motifs. However, this hTRT clone did not encode a contiguousportion of a TRT because RT motifs B′, C, D, and E, were contained in adifferent open reading frame than the more N-terminal RT motifs. Inaddition, the distance between RT motifs A and B was substantiallyshorter than that of the three previously known (non-human) TRTs.

To isolate a full length cDNA clone, a cDNA library derived form thehuman 293 cell line (described above) which expresses high levels oftelomerase activity, was screened. A lambda cDNA library from the 293cell line was partitioned into 25 pools containing about 200,000 plaqueseach. Each pool was screened by PCR with the primer pair5′-CGGAAGAGTGTCTGGAGCAA-3′ (SEQ ID NO:551) and 5′-GGATGAAGCGGAGTCTGGA-3′(SEQ ID NO:459). Six subpools of one positive primary pool were furtherscreened by PCR using this same primer pair. For both the primary andthe secondary subpool screening, hTRT was amplified for a total of 31cycles at: 94° C., 45 seconds; 60° C., 45 seconds; and 72° C., 90seconds. As a control, RNA of the house-keeping enzyme GAPDH wasamplified using the primer pair 5′-CTCAGACACCATGGGGAAGGTGA-3′ (SEQ IDNO:552) and 5′-ATGATCTTGAGGCTGTTGTCATA-3′ (SEQ ID NO:553) for a total of16 cycles at 94° C., 45 seconds; 55° C., 45 seconds; and 72° C., 90seconds.

One hTRT positive subpool from the secondary screening was then screenedby plaque hybridization with a probe from the 5′ region of clone#712562. One phage was positively identified (designated Lambda phage25-1.1, ATCC 209024, deposited May 12, 1997). It contained anapproximately four kilobase insert, which was excised and subcloned intothe EcoRI site of pBluescript II SK+ vector (Stratagene, San Diego,Calif.) as an EcoRI fragment. This cDNA clone-containing plasmid wasdesignated pGRN121. The cDNA insert totals approximately 4kilobasepairs. The complete nucleotide sequence of the human hTRT cDNA(pGRN121) has been deposited in Genbank (accession AF015950) and theplasmid has been deposited with the ATCC (ATCC 209016, deposited May 6,1997).

B. Growth of Euplotes aediculatus

In this Example, cultures of E. aediculatus were obtained from Dr. DavidPrescott, MCDB, University of Colorado. Dr. Prescott originally isolatedthis culture from pond water, although this organism is also availablefrom the ATCC (ATCC #30859). Cultures were grown as described by Swantonet al., (Swanton et al., Chromosoma 77:203 [1980]), under non-sterileconditions, in 15-liter glass containers containing Chlorogonium as afood source. Organisms were harvested from the cultures when the densityreached approximately 10⁴ cells/ml.

C. Preparation of Nuclear Extracts

In this Example, nuclear extracts of E. aediculatus were prepared usingthe method of Lingner et al., (Lingner et al., Genes Develop., 8:1984[1994]), with minor modifications, as indicated below. Briefly, cellsgrown as described in Part B were concentrated with 15 μm Nytex filtersand cooled on ice. The cell pellet was resuspended in a final volume of110 ml TMS/PMSF/spermidine phosphate buffer. The stockTMS/PMSF/spermidine phosphate buffer was prepared by adding 0.075 gspermidine phosphate (USB) and 0.75 ml PMSF (from 100 mM stock preparedin ethanol) to 150 ml TMS. TMS comprised 10 mM Tris-acetate, 10 mMMgCl₂, 85.5752 g sucrose/liter, and 0.33297 g CaCl₂/liter, pH 7.5.

After resuspension in TMS/PMSF/spermidine phosphate buffer, 8.8 ml 10%NP-40 and 94.1 g sucrose were added and the mixture placed in asiliconized glass beaker with a stainless steel stirring rod attached toan overhead motor. The mixture was stirred until the cells werecompletely lysed (approximately 20 minutes). The mixture was thencentrifuged for 10 minutes at 7500 rpm (8950×g), at 4EC, using a BeckmanJS-13 swing-out rotor. The supernatant was removed and nuclei pellet wasresuspended in TMS/PMSF/spermidine phosphate buffer, and centrifugedagain, for 5 minutes at 7500 rpm (8950×g), at 4EC, using a Beckman JS-13swing-out rotor.

The supernatant was removed and the nuclei pellet was resuspended in abuffer comprised of 50 mM Tris-acetate, 10 mM MgCl₂, 10% glycerol, 0.1%NP-40, 0.4 M KGlu, 0.5 mM PMSF, pH 7.5, at a volume of 0.5 ml buffer per10 g of harvested cells. The resuspended nuclei were then dounced in aglass homogenizer with approximately 50 strokes, and then centrifugedfor 25 minutes at 14,000 rpm at 4° C., in an Eppendorf centrifuge. Thesupernatant containing the nuclear extract was collected, frozen inliquid nitrogen, and stored at −80° C. until used.

D. Purification of Telomerase

In this Example, nuclear extracts prepared as described in Part C wereused to purify E. aediculatus telomerase. In this purification protocol,telomerase was first enriched by chromatography on an Affi-Gel-heparincolumn, and then extensively purified by affinity purification with anantisense oligonucleotide. As the template region of telomerase RNA isaccessible to hybridization in the telomerase RNP particle, an antisenseoligonucleotide (i.e., the “affinity oligonucleotide”) was synthesizedthat was complementary to this template region as an affinity bait forthe telomerase. A biotin residue was included at the 5′ end of theoligonucleotide to immobilize it to an avidin column.

Following the binding of the telomerase to the oligonucleotide, andextensive washing, the telomerase was eluted by use of a displacementoligonucleotide. The affinity oligonucleotide included DNA bases thatwere not complementary to the telomerase RNA 5′ to thetelomerase-specific sequence. As the displacement oligonucleotide wascomplementary to the affinity oligonucleotide for its entire length, itwas able to form a more thermodynamically stable duplex than thetelomerase bound to the affinity oligonucleotide. Thus, addition of thedisplacement oligonucleotide resulted in the elution of the telomerasefrom the column.

The nuclear extracts prepared from 45 liter cultures were frozen until atotal of 34 ml of nuclear extract was collected. This corresponded to630 liters of culture (i.e., approximately 4×10⁹ cells). The nuclearextract was diluted with a buffer to 410 ml, to provide finalconcentrations of 20 mM Tris-acetate, 1 mM MgCl₂, 0.1 mM EDTA, 33 mMKGlu, 10% (vol/vol) glycerol, 1 mM dithiothreitol (DTT), and 0.5 mMphenylmethylsulfonyl fluoride (PMSF), at a pH of 7.5.

The diluted nuclear extract was applied to an Affi-Gel-heparin gelcolumn (Bio-Rad), with a 230 ml bed volume and 5 cm diameter,equilibrated in the same buffer and eluted with a 2-liter gradient from33 to 450 mM KGlu. The column was run at 4° C., at a flow rate of 1column volume/hour. Fractions of 50 mls each were collected and assayedfor telomerase activity as described in Part E. Telomerase was elutedfrom the column at approximately 170 mM KGlu. Fractions containingtelomerase (approximately 440 ml) were pooled and adjusted to 20 mMTris-acetate, 10 mM MgCl₂, 1 mM EDTA, 300 mM KGlu, 10% glycerol, 1 mMDTT, and 1% Nonidet P-40. This buffer was designated as “WB.”

To this preparation, 1.5 nmol of each of two competitor DNAoligonucleotides (5′-TAGACCTGTTAGTGTACATTTGAATTGAAGC-3′; SEQ ID NO:554and 5′-TAGACCTGTTAGGTTGGATTTGTGGCATCA-3′; SEQ ID NO:552), 50 μg yeastRNA (Sigma), and 0.3 nmol of biotin-labeled telomerase-specificoligonucleotide(5′-biotin-TAGACCTGTTA-(rmeG)₂-(rmeU)₄-(rmeG)₄-(rmeU)₄-rmeG-3′; SEQ IDNO:556), were added per ml of the pool. The 2-O-methyribonucleotides ofthe telomerase specific oligonucleotides were complementary to thetelomerase RNA; template region; the deoxyribonucleotides were notcomplementary. The inclusion of competitor, non-specific DNAoligonucleotides increased the efficiency of the purification, as theeffects of nucleic acid binding proteins and other components in themixture that would either bind to the affinity oligonucleotide or removethe telomerase from the mixture were minimized.

This material was then added to Ultralink immobilized neutravidin plus(Pierce) column material, at a volume of 60 μl of suspension per ml ofpool. The column material was pre-blocked twice for 15 minutes eachblocking, with a preparation of WB containing 0.01% Nonidet P-40, 0.5 mgBSA, 0.5 mg/ml lysozyme, 0.05 mg/ml glycogen, and 0.1 mg/ml yeast RNA.The blocking was conducted at 4° C., using a rotating wheel to block thecolumn material thoroughly. After the first blocking step, and beforethe second blocking step, the column material was centrifuged at 200×gfor 2 minutes to pellet the matrix.

The pool-column mixture was incubated for 8 minutes at 30° C., and thenfor an additional 2 hours at 4E° C., on a rotating wheel (approximately10 rpm; Labindustries) to allow binding. The pool-column mixture wasthen centrifuged 200×g for 2 minutes, and the supernatant containingunbound material was removed. The pool-column mixture was then washed.This washing process included the steps of rinsing the pool-columnmixture with WB at 4° C., washing the mixture for 15 minutes with WB at4° C., rinsing with WB, washing for 5 minutes at 30° C., with WBcontaining 0.6 M KGlu, and no Nonidet P-40, washing 5 minutes at 25° C.with WB, and finally, rinsing again with WB. The volume remaining afterthe final wash was kept small, in order to yield a ratio of buffer tocolumn material of approximately 1:1.

Telomerase was eluted from the column material by adding 1 nmol ofdisplacement deoxyoligonucleotide (5′-CA₄C₄A₄C₂TA₂CAG₂TCTA-3′; SEQ IDNO:557), per ml of column material and incubating at 25° C. for 30minutes. The material was centrifuged for 2 minutes at 14,000 rpm in amicrocentrifuge (Eppendorf), and the eluate collected. The elutionprocedure was repeated twice more, using fresh displacementoligonucleotide each time. As mentioned above, because the displacementoligonucleotide was complementary to the affinity oligonucleotide, itformed a more thermodynamically stable complex with the affinityoligonucleotide than P-40. Thus, addition of the displacementoligonucleotide to an affinity-bound telomerase resulted in efficientelution of telomerase under native conditions. The telomerase appearedto be approximately 50% pure at this stage, as judged by analysis on aprotein gel. The affinity purification of telomerase and elution with adisplacement oligonucleotide is shown in FIG. 26 (panels A and B,respectively). In this Figure, the 2′-O-methyl sugars of the affinityoligonucleotide are indicated by the bold line. The black and shadedoval shapes in this figure are intended to represent graphically theprotein subunits of the present invention.

The protein concentrations of the extract and material obtainedfollowing Affi-Gel-heparin column chromatography were determined usingthe method of Bradford (Bradford, Anal. Biochem., 72:248 [1976]), usingBSA as the standard. Only a fraction of the telomerase preparation wasfurther purified on a glycerol gradient.

The sedimentation coefficient of telomerase was determined by glycerolgradient centrifugation, as described in Part I.

Table 5 below is a purification table for telomerase purified accordingto the methods of this Example. The telomerase was enriched 12-fold innuclear extracts, as compared to whole cell extracts, with a recovery of80%; 85% of telomerase was solubilized from nuclei upon extraction.

TABLE 5 Purification of Telomerase Telomer- Telomerase/ Protein ase(pmol Protein/pmol Recovery Purification Fraction (mg) of RNP) of RNP/mg(%) Factor Nuclear 2020 1720 0.9 100   1 Extract Heparin 125 1040 8.3 60 10 Affinity 0.3**  680 2270 40 2670 Glyc- NA* NA* NA* 25 NA* erol Gra-dient *NA = Not available **This value was calculated from the measuredamount of telomerase (680 pmol), by assuming a purity of 50% (based on aprotein gel).

E. Telomerase Activity

At each step in the purification of telomerase, the preparation wasanalyzed by three separate assays, one of which was activity, asdescribed in this Example. In general, telomerase assays were done in 40μl containing 0.003-0.3 μl of nuclear extract, 50 mM Tris-Cl (pH 7.5),50 mM KGlu, 10 mM MgCl₂, 1 mM DTT, 125 μM dTTP, 125 μM dGTP, andapproximately 0.2 pmoles of 5′-³²P-labelled oligonucleotide substrate(i.e., approximately 400,000 cpm). Oligonucleotide primers wereheat-denatured prior to their addition to the reaction mixture.Reactions were assembled on ice and incubated for 30 minutes at 25EC.The reactions were stopped by addition of 200 μl of 10 mM Tris-Cl (pH7.5), 15 mM EDTA, 0.6% SDS, and 0.05 mg/ml proteinase K, and incubatedfor at least 30 minutes at 45EC. After ethanol precipitation, theproducts were analyzed on denaturing 8% PAGE gels, as known in the art(See e.g., Sambrook et al., 1989).

F. Quantitation of Telomerase Activity

In this Example, quantitation of telomerase activity through thepurification procedure is described. Quantitation was accomplished byassaying the elongation of oligonucleotide primers in the presence ofdGTP and [α-³²P]dTTP. Briefly, 1 μM 5′-(G₄T₄)₂-3′ oligonucleotide wasextended in a 20 μl reaction mixture in the presence of 2 μl of[α-³²P]dTTP (10 mCi/ml, 400 Ci/mmol; 1 Ci=37 GBq), and 125 μM dGTP asdescribed (Lingner et al., Genes Develop., 8:1984 [1994]) and loadedonto an 8% PAGE sequencing gel as described.

The results of this study are shown in FIG. 28. In lane 1, there is notelomerase present (i.e., a negative control); lanes 2, 5, 8, and 11contained 0.14 fmol telomerase; lanes 3, 6, 9, and 12 contained 0.42fmol telomerase; and lanes 4, 7, 10, and 13 contained 1.3 fmoltelomerase. Activity was quantitation using a PhosphorImager (MolecularDynamics) using the manufacturer's instructions. It was determined thatunder these conditions, 1 fmol of affinity-purified telomeraseincorporated 21 fmol of dTTP in 30 minutes.

As shown in FIG. 28, the specific activity of the telomerase did notchange significantly through the purification procedure.Affinity-purified telomerase was fully active. However, it wasdetermined that at high concentrations, an inhibitory activity wasdetected and the activity of crude extracts was not linear. Thus, in theassay shown in FIG. 28, the crude extract was diluted 700-7000-fold.Upon purification, this inhibitory activity was removed and noinhibitory effect was detected in the purified telomerase preparations,even at high enzyme concentrations.

G. Gel Electrophoresis and Northern Blots

As stated in Part E, at each step in the purification of telomerase, thepreparation was analyzed by three separate assays. This Exampledescribes the gel electrophoresis and blotting procedures used toquantify telomerase RNA present in fractions and analyze the integrityof the telomerase ribonucleoprotein particle.

i) Denaturing Gels and Northern Blots

In this Example, synthetic T7-transcribed telomerase RNA of knownconcentration served as the standard. Throughout this investigation, theRNA component was used as a measure of telomerase.

A construct for phage T7 RNA polymerase transcription of E. aediculatustelomerase RNA was produced, using (PCR). The telomerase RNA gene wasamplified with primers that annealed to either end of the gene. Theprimer that annealed at the 5′ end also encoded a hammerhead ribozymesequence to generate the natural 5′ end upon cleavage of the transcribedRNA, a T7-promoter sequence, and an EcoRI site for subcloning. Thesequence of this 5′ primer was5′-GCGGGAATTCTAATACGACTCACTATAGGGAAGAAACTCTGATGAGGCCGAAAGGCCGAAACTCCACGAAAGTGGAGTAAGTTTCTCGATAATTGATCTGTAG-3′(SEQ ID NO:558). The 3′ primer included an EarI site for termination oftranscription at the natural 3′ end, and a BamHI site for cloning. Thesequence of this 3′ primer was5′-CGGGGATCCTCTTCAAAAGATGAGAGGACAGCAAAC-3′ (SEQ ID NO:559). The PCRamplification product was cleaved with EcoRI and BamHI, and subclonedinto the respective sites of pUC19 (NEB), to give “pEaT7.” Thecorrectness of this insert was confirmed by DNA sequencing. T7transcription was performed as described by Zaug et al., Biochemistry33:14935 [1994], with EarI-linearized plasmid. RNA was gel-purified andthe concentration was determined (an A₂₆₀ of 1=40 μg/ml). This RNA wasused as a standard to determine the telomerase RNA present in variouspreparations of telomerase.

The signal of hybridization was proportional to the amount of telomeraseRNA, and the derived RNA concentrations were consistent with, butslightly higher than those obtained by native gel electrophoresis.Comparison of the amount of whole telomerase RNA in whole cell RNA toserial dilutions of known T7 RNA transcript concentrations indicatedthat each E. aediculatus cell contained approximately 300,000 telomerasemolecules.

Visualization of the telomerase was accomplished by Northern blothybridization to its RNA component, using methods as described (Lingeret al., Genes Develop., 8:1984 [1994]). Briefly, RNA (less than or equalto 0.5 μg/lane) was resolved on an 8% PAGE and electroblotted onto aHybond-N membrane (Amersham), as known in the art (see e.g., Sambrook etal., 1989). The blot was hybridized overnight in 10 ml of 4×SSC,10×Denhardt's solution, 0.1% SDS, and 50 μg/ml denatured herring spermDNA. After pre-hybridizing for 3 hours, 2×10⁶ cpm probe/ml hybridizationsolution was added. The randomly labelled probe was a PCR-product thatcovered the entire telomerase RNA gene. The blot was washed with severalbuffer changes for 30 minutes in 2×SSC, 0.1% SDS, and then washed for 1hour in 0.1×SSC and 0.1% SDS at 45° C.

ii) Native Gels and Northern Blots

In this experiment, the purified telomerase preparation was run onnative (i.e., non-denaturing) gels of 3.5% polyacrylamide and 0.33%agarose, as known in the art and described (Lamond and Sproat, [1994],supra). The telomerase comigrated approximately with the xylene cyanoldye.

The native gel results indicated that telomerase was maintained as anRNP throughout the purification protocol. FIG. 27 is a photograph of aNorthern blot showing the mobility of the telomerase in differentfractions on a non-denaturing gel as well as in vitro transcribedtelomerase. In this figure, lane 1 contained 1.5 fmol telomerase RNA,lane 2 contained 4.6 fmol telomerase RNA, lane 3 contained 14 fmoltelomerase RNA, lane 4 contained 41 fmol telomerase RNA, lane 5contained nuclear extract (42 fmol telomerase), lane 6 containedAffi-Gel-heparin-purified telomerase (47 fmol telomerase), lane 7contained affinity-purified telomerase (68 fmol), and lane 8 containedglycerol gradient-purified telomerase (35 fmol).

As shown in FIG. 27, in nuclear extracts, the telomerase was assembledinto an RNP particle that migrated slower than unassembled telomeraseRNA. Less than 1% free RNA was detected by this method. However, aslower migrating telomerase RNP complex was also sometimes detected inextracts. Upon purification on the Affi-Gel-heparin column, thetelomerase RNP particle did not change in mobility (FIG. 27, lane 6).However, upon affinity purification the mobility of the RNA particleslightly increased (FIG. 27, lane 7), perhaps indicating that a proteinsubunit or fragment had been lost. On glycerol gradients, theaffinity-purified telomerase did not change in size, but approximately2% free telomerase RNA was detectable (FIG. 27, lane 8), suggesting thata small amount of disassembly of the RNP particle had occurred.

H. Telomerase Protein Composition

In this Example, the analysis of the purified telomerase proteincomposition are described.

Glycerol gradient fractions obtained as described in Part D, wereseparated on a 4-20% polyacrylamide gel (Novex). Followingelectrophoresis, the gel was stained with Coomassie brilliant blue. FIG.29 shows a photograph of the gel. Lanes 1 and 2 contained molecular massmarkers (Pharmacia) as indicated on the left side of the gel shown inFIG. 29. Lanes 3-5 contained glycerol gradient fraction pools asindicated on the top of the gel (i.e., lane 3 contained fractions 9-14,lane 4 contained fractions 15-22, and lane 5 contained fractions 23-32).Lane 4 contained the pool with 1 pmol of telomerase RNA. In lanes 6-9BSA standards were run at concentrations indicated at the top of the gelin FIG. 29 (i.e., lane 6 contained 0.5 pmol BSA, lane 7 contained 1.5pmol BSA, lane 8 contained 4.5 BSA, and lane 9 contained 15 pmol BSA).

As shown in FIG. 29, polypeptides with molecular masses of 120 and 43kDa co-purified with the telomerase. The 43 kDa polypeptide was observedas a doublet. It was noted that the polypeptide of approximately 43 kDain lane 3 migrated differently than the doublet in lane 4; it may be anunrelated protein. The 120 kDa and 43 kDa doublet each stained withCoomassie brilliant blue at approximately the level of 1 pmol, whencompared with BSA standards. Because this fraction contained 1 pmol oftelomerase RNA, all of which was assembled into an RNP particle (See,FIG. 27, lane 8), there appear to be two polypeptide subunits that arestoichiometric with the telomerase RNA. However, it is also possiblethat the two proteins around 43 kDa are separate enzyme subunits.

Affinity-purified telomerase that was not subjected to fractionation ona glycerol gradient contained additional polypeptides with apparentmolecular masses of 35 and 37 kDa, respectively. This latter fractionwas estimated to be at least 50% pure. However, the 35 kDa and 37 kDapolypeptides that were present in the affinity-purified material werenot reproducibly separated by glycerol gradient centrifugation. Thesepolypeptides may be contaminants, as they were not visible in allactivity-containing preparations.

I. Sedimentation Coefficient

The sedimentation coefficient for telomerase was determined by glycerolgradient centrifugation. In this Example, nuclear extract andaffinity-purified telomerase were fractionated on 15-40% glycerolgradients containing 20 mM Tris-acetate, with 1 mM MgCl₂, 0.1 mM EDTA,300 mM KGlu, and 1 mM DTT, at pH 7.5. Glycerol gradients were poured in5 ml (13×51 mm) tubes, and centrifuged using an SW55Ti rotor (Beckman)at 55,000 rpm for 14 hours at 4° C.

Marker proteins were run in a parallel gradient and had a sedimentationcoefficient of 7.6 S for alcohol dehydrogenase (ADH), 113 S forcatalase, 17.3 S for apoferritin, and 19.3 S for thyroglobulin. Thetelomerase peak was identified by native gel electrophoresis of gradientfractions followed by blot hybridization to its RNA component.

FIG. 30 is a graph showing the sedimentation coefficient for telomerase.As shown in this Figure, affinity-purified telomerase co-sedimented withcatalase at 11.5 S, while telomerase in nuclear extracts sedimentedslightly faster, peaking around 12.5 S. Therefore, consistent with themobility of the enzyme in native gels, purified telomerase appears tohave lost a proteolytic fragment or a loosely associated subunit.

The calculated molecular mass for telomerase, if it is assumed toconsist of one 120 kDa protein subunit, one 43 kDa subunit, and one RNAsubunit of 66 kDa, adds up to a total of 229 kDa. This is in closeagreement with the 232 kDa molecular mass of catalase. However, thesedimentation coefficient is a function of the molecular mass, as wellas the partial specific volume and the frictional coefficient of themolecule, both of which are unknown for the Euplotes telomerase RNP.

J. Substrate Utilization

In this Example, the substrate requirements of Euplotes telomerase wereinvestigated. One simple model for DNA end replication predicts thatafter semi-conservative DNA replication, telomerase extendsdouble-stranded, blunt-ended DNA molecules. In a variation of thismodel, a single-stranded 3′ end is created by a helicase or nucleaseafter replication. This 3′ end is then used by telomerase for bindingand extension.

To determine whether telomerase is capable of elongating blunt-endedmolecules, model hairpins were synthesized with telomeric repeatspositioned at their 3′ ends. These primer substrates were gel-purified,5′-end labelled with polynucleotide kinase, heated at 0.4 μM to 80° C.for 5 minutes, and then slowly cooled to room temperature in a heatingblock, to allow renaturation and helix formation of the hairpins.Substrate mobility on a non-denaturing gel indicated that very efficienthairpin formation was present, as compared to dimerization.

Assays were performed with unlabelled 125 μM dGTP, 125 μM dTTP, and 0.02μM 5′-end-labelled primer (5′-³²P-labelled oligonucleotide substrate) in10 μl reaction mixtures that contained 20 mM Tris-acetate, with 10 mMMgCl₂, 50 mM KGlu, and 1 mM DTT, at pH 7.5. These mixtures wereincubated at 25° C. for 30 minutes. Reactions were stopped by addingformamide loading buffer (i.e., TBE, formamide, bromthymol blue, andcyanol, Sambrook, 1989, supra).

Primers were incubated without telomerase (“−”), with 5.9 fmol ofaffinity-purified telomerase (“+”), or with 17.6 fmol ofaffinity-purified telomerase (“+++”). Affinity-purified telomerase usedin this assay was dialyzed with a membrane having a molecular cut-off of100 kDa, in order to remove the displacement oligonucleotide. Reactionproducts were separated on an 8% PAGE/urea gel containing 36% formamide,to denature the hairpins. The sequences of the primers used in thisstudy, as well as their lane assignments are shown in Table 6.

TABLE 6 Primer Sequences Lane Primer Sequence: (5′ to 3′) SEQ ID NO: 1-3C₄(A₄C₄)₃CACA(G₄T₄)₃G₄ 560 4-6 C₂(A₄C₄)₃CACA(G₄T₄)₃G₄ 561 7-9(A₄C₄)₃CACA(G₄T₄)₃G₄ 562 10-12 A₂C₄(A₄C₄)₂CACA(G₄T₄)₃G₄ 563 13-15C₄(A₄C₄)₂CACA(G₄T₄)₃ 564 16-18 (A₄C₄)₃CACA(G₄T₄)₃ 565 19-21A₂C₄(A₄C₄)₂CACA(G₄T₄)₃ 566 22-24 C₄(A₄C₄)₂CACA(G₄T₄)₃ 564 25-27C₂(A₄C₄)₂CACA(G₄T₄)₃ 567 28-30 (A₄C₄)₂CACA(G₄T₄)₃ 568

The gel results are shown in FIG. 31. Lanes 1-15 contained substrateswith telomeric repeats ending with four G residues. Lanes 16-30contained substrates with telomeric repeats ending with four T residues.The putative alignment on the telomerase RNA template is indicated inFIG. 32. It was assumed that the primer sets anneal at two verydifferent positions in the template shown in FIG. 32 (i.e., Panel A andPanel B, respectively). This may have affected their binding and/orelongation rate.

FIG. 33 shows a lighter exposure of lanes 25-30 in FIG. 31. The lighterexposure of FIG. 33 was taken to permit visualization of the nucleotidesthat are added and the positions of pausing in elongated products.Percent of substrate elongated for the third lane in each set wasquantified on a PhosphorImager, as indicated on the bottom of FIG. 31.

The substrate efficiencies for these hairpins were compared withdouble-stranded telomere-like substrates with overhangs of differinglengths. A model substrate that ended with four G residues (see lanes1-15 of FIG. 31) was not elongated when it was blunt ended (see lanes1-3). However, slight extension was observed with an overhang length oftwo bases; elongation became efficient when the overhang was at least 4bases in length. The telomerase acted in a similar manner with adouble-stranded substrate that ended with four T residues, with a 6-baseoverhang required for highly efficient elongation. In FIG. 31, the faintbands below the primers in lanes 10-15 that are independent oftelomerase represent shorter oligonucleotides in the primerpreparations.

The lighter exposure of lanes 25-30 in FIG. 33 shows a ladder ofelongated products, with the darkest bands correlating with the putative5′ boundary of the template (as described by Lingner et al., GenesDevelop., 8:1984 [1994]). The abundance of products that correspond toother positions in the template suggested that pausing and/ordissociation occurs at sites other than the site of translocation withthe purified telomerase.

As shown in FIG. 31, double-stranded, blunt-ended oligonucleotides werenot substrates for telomerase. To determine whether these moleculeswould bind to telomerase, a competition experiment was performed. Inthis experiment, 2 nM of 5′-end labeled substrate with the sequence(G₄T₄)₂ (SEQ ID NO:114), or a hairpin substrate with a six base overhangwere extended with 0.125 nM telomerase (FIG. 31, lanes 25-27). Althoughthe same unlabeled oligonucleotide substrates competed efficiently withlabeled substrate for extension, no reduction of activity was observedwhen the double-stranded blunt-ended hairpin oligonucleotides were usedas competitors, even in the presence of 100-fold excess hairpins.

These results indicated that double-stranded, blunt-endedoligonucleotides cannot bind to telomerase at the concentrations andconditions tested in this Example. Rather, a single-stranded 3′ end isrequired for binding. It is likely that this 3′ end is required to basepair with the telomerase RNA template.

K. Cloning & Sequencing of the 123 kDa Polypeptide

In this Example, the cloning of the 123 kDa polypeptide of Euplotestelomerase (i.e., the 123 kDa protein subunit) is described. In thisstudy, an internal fragment of the telomerase gene was amplified by PCR,with oligonucleotide primers designed to match peptide sequences thatwere obtained from the purified polypeptide obtained in Part D, above.The polypeptide sequence was determined using the nanoES tandem massspectroscopy methods known in the art and described by Calvio et al.,RNA 1:724-733 [1995]. The oligonucleotide primers used in this Examplehad the following sequences, with positions that were degenerate shownin parentheses - - -5′-TCT(G/A)AA(G/A)TA(G/A)TG(T/G/A)GT(G/A/T/C)A(T/G/A)(G/A)TT(G/A)TTCAT-3′(SEQ ID NO:569), and5′-GCGGATCCATGAA(T/C)CC(A/T)GA(G/A)AA(T/C)CC(A/T)AA(T/C)GT-3′ (SEQ IDNO:570).

A 50 μl reaction contained 0.2 mM dNTPs, 0.15 μg E. aediculatuschromosomal DNA, 0.5 μl Taq (Boehringer-Mannheim), 0.8 μg of eachprimer, and 1×reaction buffer (Boehringer-Mannheim). The reaction wasincubated in a thermocycler (Perkin-Elmer), using the following—5minutes at 95° C., followed by 30 cycles of 1 minute at 94° C., 1 minuteat 52° C., and 2 minutes at 72° C. The reaction was completed by a 10minute incubation at 72EC.

A genomic DNA library was prepared from the chromosomal E. aediculatusDNA by cloning blunt-ended DNA into the SmaI site of pCR-Script plasmidvector FIG. 14 (Stratagene). This library was screened by colonyhybridization, with the radiolabelled, gel-purified PCR product. PlasmidDNA of positive clones was prepared and sequenced by the dideoxy method(Sanger et al., Proc. Natl. Acad. Sci., 74:5463 [1977]) or manually,through use of an automated sequencer (ABI). The DNA sequence of thegene encoding this polypeptide is shown in FIG. 13. The start codon inthis sequence inferred from the DNA sequence, is located at nucleotideposition 101, and the open reading frame ends at position 3193. Thegenetic code of Euplotes differs from other organisms in that the “UGA”codon encodes a cysteine residue. The amino acid sequence of thepolypeptide inferred from the DNA sequence is shown in FIG. 14, andassumes that no unusual amino acids are inserted during translation andno post-translational modification occurs.

L. Cloning & Sequencing of the 43 kDa Polypeptide

In this Example, the cloning of the 43 kDa polypeptide of telomerase(i.e., the 43 kDa protein subunit) is described. In this study, aninternal fragment of the corresponding telomerase gene was amplified byPCR, with oligonucleotide primers designed to match peptide sequencesthat were obtained from the purified polypeptide obtained in Part D,above. The polypeptide sequence was determined using the nanoES tandemmass spectroscopy methods known in the art and described by Calvio etat., supra. The oligonucleotide primers used in this Example had thefollowing sequences - - -5′-NNNGTNAC(C/T/A)GG(C/T/A)AT(C/T/A)AA(C/T)AA-3′ (SEQ ID NO :571), and5′-(T/G/A)GC(T/G/A)GT(C/T)TC(T/C)TG(G/A)TC(G/A)TT(G/A)TA-3′ (SEQ IDNO:572). In this sequence, “N” indicates the presence of any of the fournucleotides (i.e., A, T, G, or C).

The PCR was performed as described in Part K.

A genomic DNA library was prepared and screened as described in Part K.The DNA sequence of the gene encoding this polypeptide is shown in FIG.34. Three potential reading frames are shown for this sequence, as shownin FIG. 35. For clarity, the amino acid sequence is indicated below thenucleotide sequence in all three reading frames. These reading framesare designated as “a,” “b,” and “c”. A possible start codon is encodedat nucleotide position 84 in reading frame “c.” The coding region couldend at position 1501 in reading frame “b.” Early stop codons, indicatedby asterisks in this figure, occur in all three reading frames betweennucleotide position 337-350.

Further downstream, the protein sequence appears to be encoded bydifferent reading frames, as none of the three frames is uninterruptedby stop codons. Furthermore, peptide sequences from purified protein areencoded in all three frames. Therefore, this gene appears to containintervening sequences, or in the alternative, the RNA is edited. Otherpossibilities include ribosomal frame-shifting or sequence errors.However, the homology to the La-protein sequence remains of significantinterest. Again, in Euplotes, the “UGA” codon encodes a cysteineresidue.

M. Amino Acid and Nucleic Acid Comparisons

In this Example, comparisons between various reported sequences and thesequences of the 123 kDa and 43 kDa telomerase subunit polypeptides weremade.

i) Comparisons with the 123 kDa E. aediculatus Telomerase Subunit

The amino acid sequence of the 123 kDa Euplotes aediculatus polypeptidewas compared with the sequence of the 80 kDa telomerase protein subunitof Tetrahymena thermophila (GenBank accession #U25641) to investigatetheir similarity. The nucleotide sequence as obtained from GenBankencoding this protein is shown in FIG. 42. The amino acid sequence ofthis protein as obtained from GenBank is shown in FIG. 43. The sequencecomparison between the 123 kDa E. aedicutlatus and 80 kDa T. thermophilais shown in FIG. 36. In this figure, the E. aediculatus sequence is theupper sequence, while the T. thermophila sequence is the lower sequence.The observed identity was determined to be approximately 19%, while thepercent similarity was approximately 45%, values similar to what wouldbe observed with any random protein sequence. In FIGS. 36-39, identitiesare indicated by vertical bars, while single dots between the sequencesindicate somewhat similar amino acids, and double dots between thesequences indicate more similar amino acids.

The amino acid sequence of the 123 kDa Euplotes aediculatus polypeptidewas also compared with the sequence of the 95 kDa telomerase proteinsubunit of Tetrahymena thermophila (GenBank accession #U25642), toinvestigate their similarity. The nucleotide sequence as obtained fromGenBank encoding this protein is shown in FIG. 44. The amino acidsequence of this protein as obtained from GenBank is shown in FIG. 45.This sequence comparison is shown in FIG. 37. In this figure, the E.aediculatus sequence is the upper sequence), while the T. thermophilasequence is the lower sequence. The observed identity was determined tobe approximately 20%, while the percent similarity was approximately43%, values similar to what would be observed with any random proteinsequence.

Significantly, the amino acid sequence of the 123 kDa E. aediculatuspolypeptide contains the five motifs characteristic of reversetranscriptases. The 123 kDa polypeptide was also compared with thepolymerase domains of various reverse transcriptases. FIG. 40 shows thealignment of the 123 kDa polypeptide with the putative yeast homolog(L8543.12 or ESTp). The amino acid sequence of L8543.12 obtained fromGenBank is shown in FIG. 46.

Four motifs (A, B, C, and D) were included in this comparison. In thisFIG. 40, highly conserved residues are indicated by white letters on ablack background. Residues of the E. aediculatus sequences that areconserved in the other sequence are indicated in bold; the “h” indicatesthe presence of a hydrophobic amino acid. The numerals located betweenamino acid residues of the motifs indicates the length of gaps in thesequences. For example, the “100” shown between motifs A and B reflectsa 100 amino acid gap in the sequence between the motifs.

As noted above, Genbank searches identified a yeast protein (Genbankaccession #u20618), and gene L8543.12 (Est2) containing or encodingamino acid sequence that shows some homology to the E. aediculatus 123kDa telomerase subunit. Based on the observations that both proteinscontain reverse transcriptase motifs in their C-terminal regions; bothproteins share similarity in regions outside the reverse transcriptasemotif; the proteins are similarly basic (pI=10.1 for E. aediculatus andpI=10.0 for the yeast); and both proteins are large (123 kDa for E.aediculatus and 103 kDa for the yeast), these sequences comprise thecatalytic core of their respective telomerases. It was contemplatedbased on this observation of homology in two phylogenetically distinctorganisms as E. aediculatus and yeast, that human telomerase wouldcontain a protein that has the same characteristics (i.e., reversetranscriptase motifs, is basic, and large [>100 kDa]).

ii) Comparisons with the 43 kDa E. aediculatus Telomerase Subunit

The amino acid sequence of the “La-domain” of the 43 kDa Euplotesaediculatus polypeptide was compared with the sequence of the 95 kDatelomerase protein subunit of Tetrahymena thermophila (described above)to investigate their similarity. This sequence comparison is shown inFIG. 38, while the T. thermophila sequence is the lower sequence. Theobserved identity was determined to be approximately 23%, while thepercent similarity was approximately 46%, values similar to what wouldbe observed with any random protein sequence.

The amino acid sequence of the “La-domain” of the 43 kDa Euplotesaediculatus polypeptide was compared with the sequence of the 80 kDatelomerase protein subunit of Tetrahymena thermophila (described above)to investigate their similarity. This sequence comparison is shown inFIG. 39. In this figure, the E. aediculatus sequence is the uppersequence, while the T. thermophila sequence is the lower sequence. Theobserved identity was determined to be approximately 26%, while thepercent similarity was approximately 49%, values similar to what wouldbe observed with any random protein sequence.

The amino acid sequence of a domain of the 43 kDa E. aediculatuspolypeptide was also compared with La proteins from various otherorganisms. These comparisons are shown in FIG. 41. In this Figure,highly conserved residues are indicated by white letters on a blackbackground. Residues of the E. aediculatus sequences that are conservedin the other sequence are indicated in bold.

N. Identification of Telomerase Protein Subunits in Another Organism

In this Example, the sequences identified in the previous Examples abovewere used to identify the telomerase protein subunits of Oxytrichatrifallax, a ciliate that is very distantly related to E. aediculatus.Primers were chosen based on the conserved region of the E. aediculatus123 kDa polypeptide which comprised the reverse transcriptase domainmotifs. Suitable primers were synthesized and used in a PCR reactionwith total DNA from Oxytricha. The Oxytricha DNA was prepared accordingto methods known in the art. The PCR products were then cloned andsequenced using methods known in the art.

The oligonucleotide sequences used as the primers were as follows:

5′-(T/C)A(A/G)AC(T/A/C)AA(G/A)GG(T/A/C)AT(T/C)CC(C/T/A)(C/T)A(G/A)GG-3′(SEQ ID NO:573) and5′-(G/A/T)GT(G/A/T)ATNA(G/A)NA(G/A)(G/A)TA(G/A)TC(G/A)TC-3′ (SEQ IDNO:574).

Positions that were degenerate are shown in parentheses, with thealternative bases shown within the parenthesis. “N” represents any ofthe four nucleotides.

In the PCR reaction, a 50 μl reaction contained 0.2 mM dNTPs, 0.3 μgOxytricha trifallax chromosomal DNA, 1 μl Taq polymerase(Boehringer-Mannheim), 2 micromolar of each primer, 1×reaction buffer(Boehringer-Mannheim). The reaction was incubated in a thermocycler(Perkin-Elmer) under the following conditions: 5 min at 95° C., 30cycles consisting of 1 min at 94° C., 1 min at 53° C, and 1 min at 72°C., followed by a 10 min incubation at 72° C. The PCR-product wasgel-purified and sequenced by the dideoxy-method (e.g., Sanger et al.,Proc. Natl. Acad. Sci. 74, 5463-5467 (1977).

The deduced amino acid sequence of the PCR product was determined andcompared with the E. aediculatus sequence. FIG. 47 shows the alignmentof these sequences, with the O. trifallax sequence shown in the top row,and the E. aediculatus sequence shown in the bottom row. As can be seenfrom this figure, there is a great deal of homology between the O.trifallax polypeptide sequence identified in this Example with the E.aediculatus polypeptide sequence. Thus, it is clear that the sequencesidentified in the present invention are useful for the identification ofhomologous telomerase protein subunits in other eukaryotic organisms.Indeed, development of the present invention has identified homologoustelomerase sequences in multiple, diverse species, as described herein.

O. Identification of Tetrahymena Telomerase Sequences

In this Example, a Tetrahymena clone was produced that shares homologywith the Euplotes sequences, and EST2p.

This experiment utilized PCR with degenerate oligonucleotide primersdirected against conserved motifs to identify regions of homologybetween Tetrahymena, Euplotes, and EST2p sequences. The PCR method usedin this Example is a novel method designed to amplify specifically rareDNA sequences from complex mixtures. This method avoids the problem ofamplification of DNA products with the same PCR primer at both ends(i.e., single primer products) commonly encountered in PCR cloningmethods. These single primer products produce unwanted background andcan often obscure the amplification and detection of the desiredtwo-primer product. The method used in this experiment preferentiallyselects for two-primer products. In particular, one primer isbiotinylated and the other is not. After several rounds of PCRamplification, the products are purified using streptavidin magneticbeads and two primer products are specifically eluted using heatdenaturation. This method finds use in settings other than theexperiments described in this Example. Indeed, this method finds use inapplication in which it is desired to specifically amplify rare DNAsequences, including the preliminary steps in cloning methods such as 5′and 3′RACE, and any method that uses degenerate primers in PCR.

A first PCR run was conducted using Tetrahymena template macronuclearDNA isolated using methods known in the art, and the 24-mer forwardprimer with the sequence5′biotin-GCCTATTT(TC)TT(TC)TA(TC)(GATC)(GATC)(GATC)AC(GATC)GA-3′ (SEQ IDNO:575) designated as “K231,” corresponding to the FFYXTE SEQ ID NO:360region, and the 23-mer reverse primer with the sequence5′-CCAGATAT(GATC)A (TGA)(GATC)A(AG)(AG)AA(AG)TC(AG)TC-3′ (SEQ IDNO:576), designated as “K220,” corresponding to the DDFL(FIL)I (SEQ IDNO:577) region. This PCR reaction contained 2.5 μl DNA (50 ng), 4 μl ofeach primer (20 μM), 3 μl 10×PCR buffer, 3 μl 10×dNTPs, 2 μl Mg, 0.3 μlTaq, and 11.2 μl dH₂O. The mixture was cycled for 8 cycles of 94° C. for45 seconds, 37° C. for 45 seconds, and 72° C. for 1 minute.

This PCR reaction was bound to 200 μl streptavidin magnetic beads,washed with 200 μl TE, resuspended in 20 μl dH₂O and then heat-denaturedby boiling at 100° C. for 2 minutes. The beads were pulled down and theeluate removed. Then, 2.5 μl of this eluate was subsequently reamplifiedusing the above conditions, with the exception being that 0.3 μl ofα-³²P dATP was included, and the PCR was carried out for 33 cycles. Thisreaction was run a 5% denaturing polyacrylamide gel, and the appropriateregion was cut out of the gel. These products were then reamplified foran additional 34 cycles, under the conditions listed above, with theexception being that a 42° C. annealing temperature was used.

A second PCR run was conducted using Tetrahymena macronuclear DNAtemplate isolated using methods known in the art, and the 23-mer forwardprimer with the sequence 5′-ACAATG(CA)G(GATC)(TCA)T(GATC)(TCA)T(GATC)CC(GATC)AA(AG)AA-3′ (SEQ ID NO:578), designated as “K228,” correspondingto the region R(LI)(LI)PKK (SEQ ID NO:579), and a reverse primer withthe sequence5′-ACGAATC(GT)(GATC)G(TAG)AT(GATC)(GC)(TA)(AG)TC(AG)TA(AG)CA 3′ (SEQ IDNO:580), designated “K224,” corresponding to the CYDSIPR (SEQ ID NO:581)region. This PCR reaction contained 2.5 μl DNA (50 ng), 4 μl of eachprimer (20 μM), 3 μl 10×PCR buffer, 3 μl 10×dNTPs, 2 μl Mg, 0.3 μl α-³²PdATP, 0.3 μl Taq, and 10.9 μl dH₂O. This reaction was run on a 5%denaturing polyacrylamide gel, and the appropriate region was cut out ofthe gel. These products were reamplified for an additional 34 cycles,under the conditions listed above, with the exception being that a 42°C. annealing temperature was used.

Ten μl of the reaction product from run 1 were bound tostreptavidin-coated magnetic beads in 200 μl TE. The beads were washedwith 200 μl TE, and then resuspended in 20 μl of dH₂O, heat denatured,and the eluate was removed. The reaction product from run 2 was thenadded to the beads and diluted with 30 μl 0.5×SSC. The mixture washeated from 94° C. to 50° C. The eluate was removed and the beads werewashed three times in 0.5×SSC at 55EC. The beads were then resuspendedin 20 μl dH₂O, heat denatured, and the eluate was removed, designated as“round 1 eluate” and saved.

To isolate the Tetrahymena band, the round 1 eluate was reamplified withthe forward primer K228 and reverse primer K227 with the sequence5′-CAATTCTC(AG)TA(AG)CA(GATC)(CG)(TA)(CT)TT(AGT)AT(GA)TC-3′ (SEQ IDNO:582), corresponding to the DIKSCYD (SEQ ID NO:583) region. The PCRreactions were conducted as described above. The reaction products wererun on a 5% polyacrylamide gel; the band corresponding to approximately295 nucleotides was cut from the gel and sequenced.

The clone designated as 168-3 was sequenced. The DNA sequence (includingthe primer sequences) was found to be:

GATTACTCCCGAAGAAAGGATCTTTCCGTCCAATCATGACTTTCTTAAGAAAGGACAAGCAAAAAAATATTAAGTTAAATCTAAATTAAATTCTAATGGATAGCCAACTTGTGTTTAGGAATTTAAAAGACATGCTGGGATAAAAGATAGGATACTCAGTCTTTGATAATAAACAAATTTCAGAAAAATTTGCCTAATTCATAGAGAAATGGAAAAATAAAGGAAGACCTCAGCTATATTATGTCACTCTAGACATAAAGACTTGCTAC(SEQ ID NO:584).

Additional sequence of this gene was obtained by PCR using one uniqueprimer designed to match the sequence from 168-3 (“K297” with thesequence 5′-GAGTGACATAATATACGTGA-3′ (SEQ ID NO:585); and the K231(FFYXTE; SEQ ID NO:360) primer. The sequence of the fragment obtainedfrom this reaction, together with 168-3 is as follows (without theprimer sequences):

AAACACAAGGAAGGAAGTCAAATATTCTATTACCGTAAACCAATATGGAAATTAGTGAGTAAATTAACTATTGTCAAAGTAAGAATTTAGTTTTCTGAAAAGAATAAATAAATGAAAAATAATTTTTATCAAAAAATTTAGCTTGAAGAGGAGAATTTGGAAAAAGTTGAAGAAAAATTGATACCAGAAGATTCATTTTAGAAATACCCTCAAGGAAAGCTAAGGATTATACCTAAAAAAGGATCTTTCCGTCCAATCATGACTTTCTTAAGAAAGGACAAGCAAAAAAATATTAAGTTAAATCTAAATTAAATTCTAATGGATAGCCAACTTGTGTTTAGGAATTTAAAAGACATGCTGGGATAAAAGATAGGATACTCAGTCTTTGATAATAAACAAATTTCAGAAAAATTTGCCTAATTCATAGAGAAATGGAAAAATAAAGGAAGACCTCAGCTATATTATGTCACTCTA(SEQ ID NO:586).

The amino acid sequence corresponding to this DNA fragment was found tobe:

KHKEGSQIFYYRKPIWKLVSKLTIVKVRIQFSEKNKQMKNNFYQKIQLEEENLEKVEEKLIPEDSFQKYPQGKLRIIPKKGSFRPIMTFLRKDKQKNIKLNLNQILMDSQLVFRNLKDMLGQKIGYSVFDNKQISEKFAQFIEKWKNKGRPQLYYVTL(SEQ ID NO:228).

This amino acid sequence was then aligned with other telomerase genes(EST2p, and Euplotes). The alignment is shown in FIG. 53. A consensussequence is also shown in this Figure.

P. Identification of Schizosaccharomyces pombe Telomerase Sequences

In this Example, the tez1 sequence of S. pombe was identified as ahomolog of the E. aediculatus p123, and S. cerevisiae Est2p.

FIG. 55 provides an overall summary of these experiments. In thisFigure, the top portion (Panel A) shows the relationship of twooverlapping genomic clones, and the 5825 bp portion that was sequenced.The region designated at “tez1⁺” is the protein coding region, with theflanking sequences indicated as well, the box underneath the 5825 bpregion is an approximately 2 kb HindIII fragment that was used to makethe tez1 disruption construct, as described below.

The bottom half of FIG. 55 (Panel B) is a “close-up” schematic of thissame region of DNA. The sequence designated as “original PCR” is theoriginal degenerate PCR fragment that was generated with a degenerateoligonucleotide primer pair designed based on Euplotes sequence motif 4(B′) and motif 5 (C), as described.

i) PCR With Degenerate Primers

PCR using degenerate primers was used to find the homolog of the E.aediculatus p123 in S. pombe. FIG. 56 shows the sequences of thedegenerate primers (designated as “poly 4” and “poly 1”) used in thisreaction. The PCR runs were conducted using the same buffer as describedin previous Examples (See e.g., Part K, above), with a 5 minute ramptime at 94° C., followed by 30 cycles of 94° C. for 30 seconds, 50° C.for 45 seconds, and 72° C. for 30 seconds, and 7 minutes at 72° C.,followed by storage at 4° C. PCR runs were conducted using variedconditions, (i.e., various concentrations of S. pombe DNA and MgCl₂concentrations). The PCR products were run on agarose gels and stainedwith ethidium bromide as described above. Several PCR runs resulted inthe production of three bands (designated as “T,” “M,” and “B”). Thesebands were re-amplified and run on gels using the same conditions asdescribed above. Four bands were observed following thisre-amplification (“T,” “M1,” “M2,” and “B”), as shown in FIG. 57. Thesefour bands were then re-amplified using the same conditions as describedabove. The third band from the top of the lane in FIG. 57 was identifiedas containing the correct sequence for a telomerase protein. The PCRproduct designated as M2 was found to show a reasonable match with othertelomerase proteins, as indicated in FIG. 58. In addition to thealignment shown, this Figure also shows the actual sequence of tez1. Inthis Figure, the asterisks indicate residues shared with all foursequences (Oxytricha “Ot”; E. aediculatus “Ea_p123”; S. cerevisiae“Sc_p103”; and M2), while the circles (i.e., dots) indicate similaramino acid residues.

ii) 3′ RT PCR

To obtain additional sequence information, 3′ and 5′ RT PCR wereconducted on the telomerase candidate identified in FIG. 58. FIG. 59provides a schematic of the 3′ RT PCR strategy used. First, cDNA wasprepared from mRNA using the oligonucleotide primer “Q^(T),” (5′-CCA GTGAGC AGA GTG ACG AGG ACT CGA GCT CAA GCT TTT TTT TTT TTT TT-3′; SEQ IDNO:587), then using this cDNA as a template for PCR with “Q_(O)” (5′-CCAGTG AGC AGA GTG ACG-3′; SEQ ID NO:588), and a primer designed based onthe original degenerated PCR reaction (i.e., “M2-T” with the sequence5′-G TGT CAT TTC TAT ATG GAA GAT TTG ATT GAT G-3′; SEQ ID NO:589). Thesecond PCR reaction (i.e., nested PCR) with “Q₁” (5′-GAG GAC TCG AGC TCAAGC-3′; SEQ ID NO:590), and another PCR primer designed with sequencederived from the original degenerate PCR reaction or “M2-T2” (5′-AC CTATCG TTT ACG AAA AAG AAA GGA TCA GTG-3′; SEQ ID NO:591). The buffers usedin this PCR were the same as described above, with amplificationconducted beginning with a ramp up of 940 for 5 min, followed by 30cycles of 94° for 30 sec, 55° C. for 30 sec, and 72° C. for 3 min,followed by 72° C. The reaction products were stored at 4° C. until use.

iii) Screening of Genomic and cDNA Libraries

After obtaining this additional sequence information, several genomicand cDNA libraries were screened to identify any libraries that containthis telomerase candidate gene. The approach used, as well as thelibraries and results are shown in FIG. 60. In this Figure, Panel Alists the libraries tested in this experiment; Panel B shows the regionsused; Panels C and D show the dot blot hybridization results obtainedwith these libraries. Positive libraries were then screened by colonyhybridization to obtain genomic and cDNA version of tez1 gene. In thisexperiment, approximately 3×10⁴ colonies from the HindIII genomiclibrary were screened and six positive clones were identified(approximately 0.01%). DNA was then prepared from two independent clones(A5 and B2). FIG. 61 shows the results obtained with theHindIII-digested A5 and B2 positive genomic clones.

In addition, cDNA REP libraries were used. Approximately 3×10⁵ colonieswere screened, and 5 positive clones were identified (0.002%). DNA wasprepared from three independent clones (2-3, 4-1, and 5-20). In laterexperiments, it was determined that clones 2-3 and 5-20 containedidentical inserts.

iv) 5′ RT PCR

As the cDNA version of gene produced to this point was not complete, 5′RT-PCR was conducted to obtain a full length clone. The strategy isschematically shown in FIG. 62. In this experiment, cDNA was preparedusing DNA oligonucleotide primer “M2-B” (5′-CAC TGA TCC TTT CTT TTT CGTAAA CGA TAG GT-3′; SEQ ID NO:592) and “M2-B2” (5′-C ATC AAT CAA ATC TTCCAT ATA GAA ATG ACA-3′; SEQ ID NO:593), designed from known regions oftez1 identified previously. An oligonucleotide linker PCR Adapt SfiIwith a phosphorylated 5′ end (“P”) (P-GGG CCG TGT TGG CCT AGT TCT CTGCTC-3′ SEQ ID NO:594; was then ligated at the 3′ end of this cDNA, andthis construct was used as the template for nested PCR. In the firstround of PCR, PCR Adapt SFI and M2-B were used as the primers; while PCRAdapt SfiI (5′-GAG GAG GAG AAG AGC AGA GAA CTA GGC CAA CAC GCC CC-3′;SEQ ID NO:595), and M2-B2 were used as primers in the second round.Nested PCR was used to increase specificity of reaction.

v) Sequence Alignments

Once the sequence of tez1 was identified, it was compared with sequencespreviously described. FIG. 63 shows the alignment of RT domains fromtelomerase catalytic subunits of S. pombe (“S.p. Tez1p”), S. cerevisiae(“S.c. Est2p”), and E. aediculatus p123 (“E.a. p123”). In this Figure,“h” indicates hydrophobic residues, while “p” indicates small polarresidues, and “c” indicates charged residues. The amino acid residuesindicated above the alignment show a known consensus RT motif of Y.Xiong and T. H. Eickbush (Y. Xiong and T. H. Eickbush, EMBO J., 9:3353-3362 [1990]). The asterisks indicate the residues that areconserved for all three proteins. “Motif O” is identified herein and inFIG. 63 as a motif specific to this telomerase subunit and not found inreverse transcriptases in general. It is therefore valuable inidentifying other amino acid sequences as telomerase catalytic subunits.

FIG. 64 shows the alignment of entire sequences from Euplotes(“Ea_p123”), S. cerevisiae (“Sc_Est2p”), and S. pombe (“Sp_Tez1p”). InPanel A, the shaded areas indicate residues shared between twosequences. In Panel B, the shaded areas indicate residues shared betweenall three sequences.

vi) Genetic Disruption of tez1

In this Example, the effects of disruption of tez1 were investigated. Astelomerase is involved in telomere maintenance, it was hypothesized thatif tez1 were indeed a telomerase component, disruption of tez1 wouldcause gradual telomere shortening.

In these experiments, homologous recombination was used to disrupt thetez1 gene in S. pombe specifically. This approach is schematicallyillustrated in FIG. 65. As indicated in FIG. 65, wild type tez1 wasreplaced with a fragment containing the ura4 or LEU2 marker.

The disruption of tez1 gene was confirmed by PCR (FIG. 66), and aSouthern blot was performed to check for telomere length. FIG. 67 showsthe Southern blot results for this experiment. Because an ApaIrestriction enzyme site is present immediately adjacent to telomericsequence in S. pombe, ApaI digestion of S. pombe genomic DNApreparations permits analysis of telomere length. Thus, DNA from S.pombe was digested with ApaI and the digestion products were run on anagarose gel and probed with a telomeric sequence-specific probe todetermine whether the telomeres of disrupted S. pombe cells wereshortened. The results are shown in FIG. 67. From these results, it wasclear that disruption of the tez1 gene caused a shortening of thetelomeres.

Q. Cloning and Characterization of Human Telomerase Protein and cDNA

In this Example, the nucleic and amino acid sequence information forhuman telomerase was determined. Partial homologous sequences were firstidentified in a BLAST search conducted using the Euplotes 123 kDapeptide and nucleic acid sequences, as well as Schizosaccharomycesprotein and corresponding cDNA (tez1) sequences. The human sequences(also referred to as “hTCP1.1”) were identified from a partial cDNAclone (clone 712562). Sequences from this clone were aligned with thesequences determined as described in previous Examples.

FIG. 1 shows the sequence alignment of the Euplotes (“p123”),Schizosaccharomyces (“tez1”), Est2p (i.e., the S. cerevisiae proteinencoded by the Est2 nucleic acid sequence, and also referred to hereinas “L8543.12”), and the human homolog identified in this comparisonsearch. FIG. 51 shows the amino acid sequence of tez1, while FIG. 52shows the DNA sequence of tez1. In FIG. 52, the introns and othernon-coding regions, are shown in lower case, while the exons (i.e.,coding regions) are shown in upper case.

As shown in the Figures, there are regions that are highly conservedamong these proteins. For example, as shown in FIG. 1, there are regionsof identity in “Motif 0,” “Motif 1,” “Motif 2,” and “Motif 3.” Theidentical amino acids are indicated with an asterisk (*), while thesimilar amino acid residues are indicated by a circle (•). Thisindicates that there are regions within the telomerase motifs that areconserved among a wide variety of eukaryotes, ranging from yeast tociliates to humans. It is contemplated that additional organisms willlikewise contain such conserved regions of sequence. FIG. 49 shows thepartial amino acid sequence of the human telomerase motifs, while FIG.50 shows the corresponding DNA sequence.

Sanger dideoxy sequencing and other methods were used, as known in theart to obtain complete sequence information of clone 712562. Some of theprimers used in the sequencing are shown in Table 7. These primers weredesigned to hybridize to the clone, based on sequence complementarity toeither plasmid backbone sequence or the sequence of the human cDNAinsert in the clone.

TABLE 7 Primers Primer Sequence SEQ ID NO: TCP1.1 GTGAAGGCACTGTTCAGCG377 TCP1.2 GTGGATGATTTCTTGTTGG 381 TCP1.3 ATGCTCCTGCGTTTGGTGG 596 TCP1.4CTGGACACTCAGCCCTTGG 382 TCP1.5 GGCAGGTGTGCTGGACACT 383 TCP1.6TTTGATGATGCTGGCGATG 384 TCP1.7 GGGGCTCGTCTTCTACAGG 385 TCP1.8CAGCAGGAGGATCTTGTAG 386 TCP1.9 TGACCCCAGGAGTGGCACG 387 TCP1.10TCAAGCTGACTCGACACCG 388 TCP1.11 CGGCGTGACAGGGCTGC 389 TCP1.12GCTGAAGGCTGAGTGTCC 390 TCP1.13 TAGTCCATGTTCACAATCG 391

From these experiments, it was determined that the EcoRI-NotI insert ofclone 712562 contains only a partial open reading frame for the humantelomerase protein, although it may encode an active fragment of thatprotein. The open reading frame in the clone encodes an approximately 63kD protein. The sequence of the longest open reading frame identified isshown in FIG. 68. The ORF begins at the ATG codon with the “met”indicated in the Figure. The poly A tail at the 3′ end of the sequenceis also shown. FIG. 69 shows a tentative, preliminary alignment oftelomerase reverse transcriptase proteins from the human sequence (humanTelomerase Core Protein 1, “Hs TCP1”), E. aediculatus p123 (“Ep p123”),S. pombe tez1 (“Sp Tez1”), S. cerevisiae EST2 (“Sc Est2”), and consensussequence. In this Figure various motifs are indicated.

To obtain a full-length clone, probing of a cDNA library and 5′-RACEwere used to obtain clones encoding portions of the previously unclonedregions. In these experiments, RACE (Rapid Amplification of cDNA Ends;See e.g., M. A. Frohman, “RACE: Rapid Amplification of cDNA Ends,” inInnis et al. (eds), PCR Protocols: A Guide to Methods and Applications[1990], pp. 28-38; and Frohman et al., Proc. Natl. Acad. Sci.,85:8998-9002 [1988]) was used to generate material for sequenceanalysis. Four such clones were generated and used to provide additional5′ sequence information (pFWRP5, 6, 19, and 20).

In addition, human cDNA libraries (inserted into lambda) were probedwith the EcoRI-NotI fragment of the clone. One lambda clone, designated“lambda 25-1.1” (ATCC accession #209024), was identified as containingcomplementary sequences. FIG. 75 shows a restriction map of this lambdaclone. The human cDNA insert from this clone was subcloned as an EcoRIrestriction fragment into the EcoRI site of commercially availablephagemid pBluescriptIISK+ (Stratagene), to create the plasmid “pGRN121,”which was deposited with the ATCC (ATCC accession #209016). Preliminaryresults indicated that plasmid pGRN121 contains the entire open readingframe (ORF) sequence encoding the human telomerase protein.

The cDNA insert of plasmid pGRN121 was sequenced using techniques knownin the art. FIG. 70 provides a restriction site and function map ofplasmid pGRN121 identified based on this preliminary work. The resultsof this preliminary sequence analysis are shown in FIG. 71. From thisanalysis, and as shown in FIG. 70, a putative start site for the codingregion was identified at approximately 50 nucleotides from the EcoRIsite (located at position 707), and the location of thetelomerase-specific motifs, “FFYVTE” (SEQ ID NO:361), “PKP,” “AYD,”“QG”, and “DD,” were identified, in addition to a putative stop site atnucleotide #3571 (See, FIG. 72, which shows the DNA and correspondingamino acid sequences for the open reading frames in the sequence (“a”,“b”, and “c”)). However, due to the preliminary nature of the earlysequencing work, the reading frames for the various motifs were foundnot to be in alignment.

Additional analysis conducted on the pGRN121 indicated that the plasmidcontained significant portions from the 5′-end of the coding sequencenot present on clone 712562. Furthermore, pGRN121 was found to contain avariant coding sequence that includes an insert of approximately 182nucleotides. This insert was found to be absent from the clone. As withthe E. aediculatus sequences, such variants can be tested in functionalassays, such as telomerase assays to detect the presence of functionaltelomerase in a sample.

Further sequence analysis resolved the cDNA sequence of pGRN121 toprovide a contiguous open reading frame that encodes a protein ofmolecular weight of approximately 127,000 daltons, and 1132 amino acidsas shown in FIG. 74. A refined map of pGRN121 based on this analysis, isprovided in FIG. 73. The results of additional sequence analysis of thehTRT cDNA are presented in FIG. 16 SEQ ID NO:1.

Example 2 Correlation of hTRT Abundance and Cell Immortality

The relative abundance of hTRT mRNA was assessed in sixtelomerase-negative mortal cell strains and six telomerase-positiveimmortal cell lines (FIG. 5). The steady state level of hTRT mRNA wassignificantly increased in immortal cell lines that had previously beenshown to have active telomerase. Lower levels of the hTRT mRNA weredetected in some telomerase-negative cell strains.

RT-PCR for hTRT, hTR, TP1 (telomerase-associated protein related toTetrahymena p80 [Harrington et al., 1997, Science 275:973; Nakayama etal., 1997, Cell 88:875]) and GAPDH (to normalize for equal amounts ofRNA template) was carried out on RNA derived from the following cells:(1) human fetal lung fibroblasts GFL, (2) human fetal skin fibroblastsGFS, (3) adult prostate stromal fibroblasts 31 YO, (4) human fetal kneesynovial fibroblasts HSF, (5) neonatal foreskin fibroblasts BJ, (6)human fetal lung fibroblasts IMR90, and immortalized cell lines (7)melanoma LOX IMVI, (8) leukemia U251, (9) NCI H23 lung carcinoma, (10)colon adenocarcinoma SW620, (11) breast tumor MCF7, (12) 293 adenovirusE1 transformed human embryonic kidney cell line.

hTRT nucleic acid was amplified from cDNA using oligonucleotide primersLt5 and Lt6 (Table 2) for a total of 31 cycles (94° C. 45s, 60° C. 45s,72° C. 90s). GAPDH was amplified using primers K136(5′-CTCAGACACCATGGGGAA GGTGA; SEQ ID NO:552) and K137(5′-ATGATCTTGAGGCTGTTGTCATA; SEQ ID NO:553) for a total of 16 cycles(94° C. 45 s, 55° C. 45 s, 72° C. 90 s). hTR was amplified using primersF3b (5′-TCTAACCCTAACTGAGAAGGGCGTAG; SEQ ID NO:597) and R3c(5′-GTTTGCTCTAGAATGAACGGTGGAAG; SEQ ID NO:598) for a total of 22 cycles(94° C. 45s, 55° C. 45 s,72° C. 90s). TP1 mRNA was amplified usingprimers TP1.1 and TP1.2 for 28 cycles (cycles the same as hTRT).Reaction products were resolved on an 8% polyacrylamide gel, stainedwith SYBR Green (Molecular Probes) and visualized by scanning on a Storm860 (Molecular Dynamics). The results, shown in FIG. 5, demonstrate thathTRT mRNA levels correlate directly with telomerase activity levels inthe cells tested.

Example 3 Characterization of an hTRT Intronic Sequence

A putative intron was first identified by PCR amplification of humangenomic DNA, as described in this example, and subsequently confirmed bysequencing the genomic clone λGφ5 (see Example 4). PCR amplification wascarried out using the forward primer TCP1.57 paired individually withthe reverse primers TCP1.46, TCP1.48, TCP1.50, TCP1.52, TCP1.54,TCP1.56, and TCP1.58 (see Table 2). The products from genomic DNA of theTCP1.57/TCP1.46, TCP1.48, TCP1.50, TCP1.52, TCP1.54, or TCP1.56amplifications were approximately 100 basepairs larger than the productsof the pGRN121 amplifications. The TCP1.57/TCP1.58 amplification was thesame on either genomic or pGRN121 DNA. This indicated the genomic DNAcontained an insertion between the sites for TCP1.58 and TCP1.50. ThePCR products of TCP1.57/TCP1.50 and TCP1.57/TCP1.52 were sequenceddirectly, without subcloning, using the primers TCP1.39, TCP1.57, andTCP1.49.

As shown below, the 104-base intronic sequence SEQ ID NO:7 is insertedin the hTRT mRNA (shown in bold) at the junction corresponding to bases274 and 275 of FIG. 16:

CCCCCCGCCGCCCCCTCCTTCCGCCAG/GTGGGCCTCCCCGGGGTCGGCGTCCGGCTGGGGTTGAGGGCGGCCGGGGGGAACCAGCGACATGCGGAGAGCAGCGCAGGCGACTCAGGGCGCTTCCCCCGCAG/GTGTCCTGCCTGAAGGAGCTGGTGGCCCGAGTGCTGCAG(SEQ ID NO:599)

The “/” indicates the splice junctions; the sequence shows good matchesto consensus 5′ and 3′ splice site sequences typical for human introns.

This intron contains motifs characteristic of a topoisomerase IIcleavage site and a NFκB binding site (see FIG. 21). These motifs are ofinterest, in part, because expression of topoisomerase II is upregulated in most tumors. It functions to relax DNA by cutting andrewinding the DNA, thus increasing expression of particular genes.Inhibitors of topoisomerase II have been shown to work as anti-tumoragents. In the case of NFκB, this transcription factor may play a rolein regulation of telomerase during terminal differentiation, such as inearly repression of telomerase during development and so is anothertarget for therapeutic intervention to regulate telomerase activity incells.

Example 4 Cloning of Lambda Phage GΦ5 and Characterization of hTRTGenomic Sequences

A. Lambda GΦ5

A human genomic DNA library was screened by PCR and hybridization toidentify a genomic clone containing hTRT RNA coding sequences. Thelibrary was a human fibroblast genomic library made using DNA from WI38lung fibroblast cells (Stratagene, Cat # 946204). In this library,partial Sau3AI fragments are ligated into the XhoI site of Lambda FIX711Vector (Stratagene), with an insert size of 9-22 kb.

The genomic library was divided into pools of 150,000 phage each, andeach pool screened by nested PCR (outer primer pair TCP1.52 & TCP1.57;inner pair TCP1.49 & TCP1.50, see Table 1). These primer pairs span aputative intron (see Example 3, supra) in the genomic DNA of hTRT andensured the PCR product was derived from a genomic source and not fromcontamination by the hTRT cDNA clone. Positive pools were furthersubdivided until a pool of 2000 phage was obtained. This pool was platedat low density and screened via hybridization with a DNA fragmentencompassing basepairs 1552-2108 of FIG. 16 (restriction sites SphI andEcoRV, respectively).

Two positive clones were isolated and rescreened via nested PCR asdescribed above; both clones were positive by PCR. One of the clones(λGΦ5) was digested with NotI, revealing an insert size of approximately20 kb. Subsequent mapping (see below) indicated the insert size was 15kb and that phage GΦ5 contains approximately 13 kb of DNA upstream fromthe start site of the cDNA sequence.

Phage GΦ5 was mapped by restriction enzyme digestion and DNA sequencing.The resulting map is shown in FIG. 7. The phage DNA was digested withNcoI and the fragments cloned into pBBS167. The resulting subclones werescreened by PCR to identify those containing sequence corresponding tothe 5′ region of the hTRT cDNA. A subclone (pGRN140) containing a 9 kbNcoI fragment (with hTRT gene sequence and 4-5 kb of lambda vectorsequence) was partially sequenced to determine the orientation of theinsert. pGRN 140 was digested using SalI to remove lambda vectorsequences, resulting in pGRN144. pGRN144 was then sequenced. The resultsof the sequencing are provided in FIG. 21. The 5′ end of the hTRT mRNAcorresponds to base 2441 of FIG. 21. As indicated in FIG. 7, two Alusequence elements are located 1700 base pairs upstream of the hTRT cDNA5′ end and provide a likely upstream limit to the promoter region ofhTRT. The sequence also reveals an intron positioned at bases 4173 inFIG. 21, 3′ to the intron described in Example 3, supra.

B. Additional Genomic Clones

In addition to the genomic clone described above, two P1 bacteriophageclones and one human BAC clone are provided as illustrative embodimentsof the invention. P1 inserts are usually 75-100 kb, and BAC inserts areusually over 100 Kb.

The P1 clones (DMPC-HFF#1-477(F6)-GS #15371 and DMPC-HEF#1-1103(H6)-GS#15372) were obtained by PCR screening of a human P1 library derivedfrom human foreskin fibroblast cells (Shepherd et al., 1994, PNAS USA91:2629) using primers TCP1.12 and UTR2 which amplify the 3′ end ofhTRT. These clones were both negative (failed to amplify) with primersthat amplify the 5′ end of hTRT.

The human BAC clone (326 E 20) was obtained with a hybridization screenof a BAC human genomic library using an 1143 bp Sph1/Xmn1 fragment ofpGRN121 (FIG. 16; bases 1552-2695) that encompasses the RT motif region.The clone is believed to include the 5′ end of the gene. The hTRTgenomic clones in this example are believed to encompass the entire hTRTgene.

Example 5 Chromosomal Location of hTRT Gene

The hTRT gene was localized to chromosome 5p by radiation hybrid mapping(Boehnke et al., 1991, Am J Hum Genet 49:1174; Walter et al., 1994,Nature Genet 7:22) using the medium resolution Stanford G3 panel of 83RH clones of the whole human genome (created at the Stanford HumanGenome Center). A human lymphoblastoid cell line (donor; rM) was exposedto 10,000 rad of x-rays and was then fused with nonirradiated hamsterrecipient cells (A3). Eighty-three independent somatic cell hybridclones were isolated, and each represents a fusion event between anirradiated donor cell and a recipient hamster cell. The panel of G3 DNAwas used for ordering markers in the region of interest as well asestablishing the distance between these markers.

The primers used for the RH mapping were TCP1.12 and UTR2 withamplification conditions of 94° C. 45 sec, 55° C. 45 sec, 72° C. 45 sec,for 45 cycles using Boehringer Mannheim Taq buffer and Perkin-Elmer Taq.The 83 pools were amplified independently and 14 (17%) scored positivefor hTRT (by appearance of a 346 bp band). The amplification resultswere submitted to Stanford RH server, which then provided the maplocation, 5p, and the closest marker, STS D5S678.

By querying the Genethon genome mapping web site, the map locationidentified a YAC that contains the STS marker D5S678: CEPH YAC780_C_(—)3 Size: 390,660 kb. This YAC also contained chromosome 17markers. This result indicated that the hTRT gene is on chromosome 5,near the telomeric end. There are increased copy numbers of 5p in anumber of tumors. Cri-du-chat syndrome also has been mapped to deletionsin this region.

Example 6 Design and Construction of Vectors for Expression of hTRTProteins and Polynucleotides

Expression of hTRT in Bacteria

The following portion of this example details the design ofhTRT-expressing bacterial and eukaryotic cell expression vectors toproduce large quantities of full-length, biologically active hTRT.Generation of biologically active hTRT protein in this manner is usefulfor telomerase reconstitution assays, assaying for telomerase activitymodulators, analysis of the activity of newly isolated species of hTRT,identifying and isolating compounds which specifically associate withhTRT, analysis of the activity of an hTRT variant protein that has beensite-specifically mutated, and as an immunogen, as a few examples.

pThioHis A/hTRT Bacterial Expression Vector

To produce large quantities of full-length hTRT, the bacterialexpression vector pThioHis A (Invitrogen, San Diego, Calif.) wasselected as an expression system. The hTRT-coding insert includesnucleotides 707 to 4776 of the hTRT insert in the plasmid pGRN121. Thisnucleotide sequence includes the complete coding sequence for the hTRTprotein.

This expression vector of the invention is designed for inducibleexpression in bacteria. The vector can be induced to express, in E.coli, high levels of a fusion protein composed of a cleavable, HIStagged thioredoxin moiety and the full length hTRT protein. The use ofthe expression system was in substantial accordance with themanufacturer's instructions. The amino acid sequence of the fusionprotein encoded by the resulting vector of the invention is shown below;(-*-) denotes an enterokinase cleavage site:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAHWCGPCKMIAPILDEIADEYQGKLTVAKLRIDHNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGDDDDK-*-VPMHELEIFEFAAASTQRCVLLRTWEALAPATPAMPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD(SEQ ID NO:600)

pGEX−2TK with hTRT Nucleotides 3272 to 4177 of pGRN121

This construct of the invention is used to produce fusion protein for,e.g., the purpose of raising polyclonal and monoclonal antibodies tohTRT protein. Fragments of hTRT can also be used for other purposes,such as to modulate telomerase activity, for example, as adominant-negative mutant or to prevent the association of a telomerasecomponent with other proteins or nucleic acids.

To produce large quantities of an hTRT protein fragment, the E. coliexpression vector pGEX-2TK (Pharmacia Biotech, Piscataway N.J.) wasselected, and used essentially according to manufacturer's instructionsto make an expression vector of the invention. The resulting constructcontains an insert derived from nucleotides 3272 to 4177 of the hTRTinsert in the plasmid pGRN121. The vector directs expression in E. coliof high levels of a fusion protein composed of glutathione-S-transferasesequence (underlined below), thrombin cleavage sequence (doubleunderlined), recognition sequence for heart muscle protein kinase(italicized), residues introduced by cloning in brackets ([GSVTK]; SEQID NO:601) and hTRT protein fragment (in bold) as shown below:

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGD (SEQ IDNO:602) VKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD LVPRGS RRASV[GSVTK]IPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASVTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD

When this fusion protein was expressed, it formed insoluble aggregates.It was treated generally as described above, in the section entitledpurification of proteins from inclusion bodies. Specifically, inducedcells were suspended in PBS (20 mM sodium phosphate, pH 7.4, 150 mMNaCl) and disrupted by sonication. NP-40 was added to 0.1%, and themixture was incubated for 30 minutes at 4° C. with gentle mixing. Theinsoluble material was collected by centrifugation at 25,000 g for 30minutes at 4° C. The insoluble material was washed once in 4M urea inPBS, collected by centrifugation, then washed again in PBS. Thecollected pellet was estimated to contain greater than 75% fusionprotein. This material was dried in a speed vacuum, then suspended inadjuvant for injection into mice and rabbits for the generation ofantibodies. Separation of the recombinant protein from the glutathioneS-transferase moiety is accomplished by site-specific proteolysis usingthrombin according to manufacturer's instructions.

pGEX-2TK with hTRT Nucleotides 2426 to 3274 of pGRN121 with HIS-8 Tag

To produce large quantities of a fragment of hTRT, another E. coliexpression vector pGEX-2TK construct was prepared. This constructcontains an insert derived from nucleotides 2426 to 3274 of the hTRTinsert in the plasmid pGRN121 and a sequence encoding eight consecutivehistidine residues (HIS-8 Tag). To insert the HIS-8 TAG, the pGEX-2TKvector with hTRT nucleotides 2426 to 3274 of pGRN 121 was linearizedwith BamH1. This opened the plasmid at the junction between theGST-thrombin-heart muscle protein kinase and the hTRT coding sequence. Adouble stranded oligonucleotide with BamH1 compatible ends was ligatedto the linearized plasmid resulting in the in-frame introduction ofeight histidine residues upstream of the hTRT sequence.

The vector directs expression in E coli of high levels of a fusionprotein composed of glutathione-S-transferase sequence (underlined);thrombin cleavage sequence (double underlined); recognition sequence forheart muscle protein kinase (italicized); a set of three and a set offive residues introduced by cloning are in brackets ([GSV] and [GSVTK]SEQ ID NO:601); eight consecutive histidines (also double underlined);and hTRT protein fragment (in bold):

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPY (SEQ IDNO:603) YIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD LVPRGS RRASV[GSV]HHHHHHHH[GSVTK]MSVYVVELLRSFFYVTETTFQKNRLFFYRPSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGI

Each of the pGEX-2TK vectors of the invention can be used to producefusion protein for the purpose of raising polyclonal and monoclonalantibodies to hTRT protein. Additionally, this fusion protein can beused to affinity purify antibodies raised to hTRT peptides that areencompassed within the fusion protein. Separation of the recombinantprotein from the glutathione S-transferase moiety can be accomplished bysite-specific proteolysis using thrombin according to manufacturer'sinstructions.

pGEX-2TK with hTRT Nucleotides 2426 to 3274 of pGRN121, no HIS-8 Tag

To produce large quantities of a fragment of hTRT, another E. coliexpression vector pGEX-2TK construct was prepared.

This construct contains an insert derived from nucleotides 2426 to 3274of the hTRT insert in the plasmid pGRN121, but without the HIS-8 tag ofthe construct described above. The vector directs expression in E coliof high levels of a fusion protein composed of glutathione-S-transferase(underlined), thrombin cleavage sequence (double underlined),recognition sequence for heart muscle protein kinase (italicized),residues introduced by cloning in brackets ([GSVTK]; SEQ ID NO:601) andhTRT protein fragment (in bold):

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGD (SEQ IDNO:604) VKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD LVPRGS RRASV[GSVTK]MSVYVVELLRSFFYVTETTFQKNRLFFYRPSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPEYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASGLFDVFLRFMCHHAVRIRGKSYVQCQGI

pGEX-2TK with hTRT Nucleotides 1625 to 2458 of pGRN121

To produce large quantities of a fragment of hTRT protein, another E.coli expression vector pGEX-2TK construct was prepared.

This construct contains an insert derived from nucleotides 1625 to 2458of the hTRT insert in the plasmid pGRN121. The vector directs expressionin E coli of high levels of a fusion protein composed ofglutathione-S-transferase, (underlined), thrombin cleavage sequence(double underlined), recognition sequence for heart muscle proteinkinase (italicized) residues introduced by cloning in brackets ([GSVTK];SEQ ID NO:601) and hTRT protein fragment (in bold):

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFP (SEQ ID NO:605)NLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGG DHPPKSDL LVPRGS[GSVTK]ATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRS

pGEX-2TK with hTRT Nucleotides 782 to 1636 of pGRN121

To produce large quantities of a fragment of hTRT protein, another E.coli expression vector pGEX-2TK construct was prepared.

This construct contains an insert derived from nucleotides 782 to 1636of the hTRT insert in the plasmid pGRN121. The vector directs expressionin E coli of high levels of a fusion protein composed ofglutathione-S-transferase, (underlined), thrombin cleavage sequence(double underlined), recognition sequence for heart muscle proteinkinase (italicized) residues introduced by cloning in brackets ([GSVTK];SEQ ID NO:601) and hTRT protein fragment (in bold):

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGD (SEQ IDNO:606) VKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD LVPRGS RRASV[GSVTK]MPRAPRCRAVRSLLSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEATTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSL

pT7FLhTRT with hTRT cDNA Lacking 5′-Non-Coding Sequence

As described above, in one embodiment, the invention provides for anhTRT that is modified in a site-specific manner to facilitate cloninginto bacterial, mammalian, yeast and insect expression vectors withoutany 5′ untranslated hTRT sequence. In some circumstances, minimizing theamount of non-protein encoding sequence allows for improved proteinproduction (yield) and increased mRNA stability. In this embodiment ofthe invention, the hTRT gene's 5′ non-coding region was removed beforecloning into a bacterial expression vector.

This was effected by engineering an additional restriction endonucleasesite just upstream (5′) to the start (ATG) codon of the hTRT codingsequence (FIG. 16). The creation of a restriction site just 5′ to thecoding region of the protein allows for efficient production of a widevariety of vectors that encode fusion proteins, such as fusion proteinscomprising labels and peptide TAGs, for immunodetection andpurification.

Specifically, the oligonucleotide 5′- CCGGCCACCCCCCATATGCCGCGCGCTCCC-3′(SEQ ID NO:607) was used as described above to modify hTRT cDNAnucleotides 779 to 781 of the hTRT cDNA (FIG. 16) from GCG to CAT. These3 nucleotides are the last nucleotides before the ATG start codon sothey do not modify the protein sequence. The change in sequence resultsin the creation of a unique NdeI restriction site in the hTRT cDNA.Single-stranded hTRT DNA was used as a DNA source for the site directedmutagenesis. The resulting plasmid was sequenced to confirm the successof the mutagenesis.

This modification allowed the construction of the following plasmid ofthe invention, designated pT7FLhTRT. The site-specifically modified hTRTsequence (addition of the NdeI restriction site) was digested with NdeIand NotI (and filled in with Klenow enzyme to generate blunt ended DNA)to generate an hTRT encoding nucleic acid fragment. The fragment wasthen cloned into a pSL3418 plasmid previously restriction digested withNdeI and SmaI (also a blunt ended cutter). pSL 3418 is a modified pAED4plasmid into which a FLAG sequence (Immunex Corp, Seattle Wash.) and anenterokinase sequence are inserted just upstream from theabove-referenced NdeI site. This plasmid, designated pT7FLhTR, allowsthe expression of full length hTRT (with a Flag-Tag at its 5′ end) in anE. coli strain expressing the T7 RNA polymerase.

Plasmids with hTRT cDNA Lacking 3′-Non-Coding Sequence

As discussed above, the invention provides for expression vectorscontaining TRT-encoding nucleic acids in which some or all non-codingsequences have been deleted. In some circumstances, minimizing theamount of non-protein encoding sequence allows for improved proteinproduction (yield) and increases mRNA stability. In this embodiment ofthe invention, the 3′ untranslated region of hTRT is deleted beforecloning into a bacterial expression plasmid.

The plasmid pGRN121, containing the full length hTRT cDNA,: as discussedabove, was first deleted of all Apa1 sites. This was followed bydeletion of the MscI-HincII hTRT restriction digest enzyme fragmentcontaining the 3′UTR. The NcoI-XbaI restriction digest fragmentcontaining the stop codon of hTRT was then inserted into the NcoI-XbaIsite of pGRN121 to make a plasmid equivalent to pGRN121, designatedpGRN124, except lacking the 3′UTR.

Bacterial Expression Vectors Using Antibiotic Selection Markers

The invention also provides for bacterial expression vectors that cancontain selection markers to confer a selectable phenotype ontransformed cells and sequences coding for episomal maintenance andreplication such that integration into the host genome is not required.For example, the marker may encode antibiotic resistance, particularlyresistance to chloramphenicol (see Harrod (1997) Nucleic Acids Res. 25:1720-1726), kanamycin, G418, bleomycin and hygromycin, to permitselection of those cells transformed with the desired DNA sequences, seefor example, Blondelet-Rouault (1997) Gene 190:315-317; and Mahan (1995)Proc Natl Acad Sci USA 92:669-673.

In one embodiment of the invention, the full length hTRT was cloned intoa modified BlueScript plasmid vector (Stratagene, San Diego, Calif.),designated pBBS235, into which a chloramphenicol antibiotic resistencegene had been inserted. The NotI fragment from pGRN124 (discussed above)containing the hTRT ORF into the NotI site of pBBS235 so that the TRTORF is in the opposite orientation of the vector's Lac promoter. Thismakes a plasmid that is suitable for mutageneis of plasmid inserts, suchas TRT nucleic acids of the invention. This plasmid construct,designated pGRN125, can be used in the methods of the inventioninvolving mutagenesis of telomerase enzyme and TRT protein codingsequences and for in vitro transcription of hTRT using the T7 promoter(and in vitro transcription of antisense hTRT using the T3 promoter).

In another embodiment of the invention, NotI restriction digestfragments from pGRN124 containing the hTRT ORF were subcloned into theNotI site of pBBS235 (described above) so the TRT ORF is in the sameorientation as the vector's Lac promoter. This makes a plasmid,designated pGRN126, that can be used for expression of full length hTRTin E. coli. The expressed product will contain 29 amino acids encoded bythe vector pBBS235, followed by 18 amino acids encoded by the 5′UTR ofhTRT, followed by the full length hTRT protein.

In a further embodiment of the invention, in vitro mutagenesis ofpGRN125 was done to convert the hTRT initiating ATG codon into a Kozakconsensus and create EcoRI and BglII restriction digest sites tofacilitate cloning into expression vectors. The oligonucleotide5′-TGCGCACGTGGGAAGCCCTGGCagatctgAattCcaCcATGCCGCGCGCTCCCCGCTG-3′ (SEQ IDNO:608) (altered nucleotides in lower case) was used in the mutagenesisprocedure. The resulting expression vector was designated pGRN127.

In another embodiment of the invention, the second Asp of the TRT “DDmotif” was converted to an alanine to create a non-functional telomerseenzyme, thus creating a mutant TRT protein for use as adominant/negative mutant. The hTRT coding sequence was mutagenized invitro using the oligonucleotide5′-CGGGACGGGCTGCTCCTGCGTTTGGTGGAcGcgTTCTTGTTGGTGACACCTCACCTCACC-3′ (SEQID NO:609) to convert the asparagine codon for residue 869 (Asp869) toan alanine (Ala) codon. This also created an MluI restriction enzymesite. The resulting expression plasmid was designated pGRN130, whichalso contains the Kozak consensus sequence as described for pGRN127.

The invention also provides a vector designed to express an antisensesequence fragment of hTRT. The pGRN126 plasmid was cut to completionwith MscI and SmaI restriction enzymes and religated to delete over 95%of the hTRT ORF. One SmaI-MscI fragment was re-inserted during theprocess to recreate CAT activity. This unpurified plasmid was thenredigested with SalI and EcoRI and the fragment containing theinitiating codon of the hTRT ORF was inserted into the SalI-EcoRI sitesof pBBS212 to make an antisense expression plasmid expressing theantisense sequence spanning the 5′UTR and 73 bases pair residues of thehTRT ORF (in mammalian cells). This plasmid was designated pGRN135.

Expression of hTRT Telomerase in Yeast

The present invention also provides hTRT-expressing yeast expressionvectors to produce large quantities of full-length, biologically activehTRT.

Pichia pastoris Expression Vector pPICZ B and Full Length hTRT

To produce large quantities of full-length, biologically active hTRT,the Picha pastoris expression vector PPICZ B (Invitrogen, San Diego,Calif.) was selected. The hTRT-coding sequence insert was derived fromnucleotides 659 to 4801 of the hTRT insert in plasmid pGRN121. Thisnucleotide sequence includes the full-length sequence encoding hTRT.This expression vector is designed for inducible expression in P.pastoris of high levels of full-length, unmodified hTRT protein.Expression is driven by a yeast promoter, but the expressed sequenceutilizes the hTRT initiation and termination codons. No exogenous codonswere introduced by the cloning. The resulting pPICZ B/hTRT vector wasused to transform the yeast.

Pichia pastoris Expression Vector hTRT-His6/pPICZ B

A second Picha pastoris expression vector of the invention derived frompPICZ B, also contains the full-length sequence encoding hTRT derivedfrom nucleotides 659 to 4801 of the hTRT insert in the plasmid pGRN121.This hTRT-His6/pPICZ B expression vector encodes full length hTRTprotein fused at its C-terminus to the Myc epitope and His6 reporter tagsequences. The hTRT stop codon has been removed and replaced by vectorsequences encoding the Myc epitope and the His6 reporter tag as well asa stop codon. This vector is designed to direct high-level inducibleexpression in yeast of the following fusion protein, which consists ofhTRT sequence (underlined), vector sequences in brackets ([L] and[NSAVD]; SEQ ID NO:610) the Myc epitope (double underlined), and theHis6 tag (italicized):

MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLV (SEQ ID NO:611)CVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD[L]EQKLISEEDL[NSAVD]HHHHHH

Expression of hTRT in Insect Cells

The present invention also provides hTRT telomerase-expressing insectcell expression vectors that produce large quantities of full-length,biologically active hTRT.

Baculovirus Expression Vector pVL1393 and Full Length hTRT

The telomerase coding sequence of interest was cloned into thebaculovirus expression vector pVL1393 (Invitrogen, San Diego, Calif.).This construct was subsequently cotransfected into Spodoptera fungupeida(sf-9) cells with linearized DNA from Autograph california nuclearpolyhedrosis virus (Baculogold-AcMNPV). The recombinant baculovirusesobtained were subsequently plaque purified and expanded followingstandard protocols.

This expression vector provides for expression in insect cells of highlevels of full-length hTRT protein. Expression is driven by abaculoviral polyhedrin gene promoter. No exogenous codons wereintroduced by the cloning.

Baculovirus Expression Vector pBlueBacHis2 B and Full Length hTRT

To produce large quantities of full-length, biologically active hTRT,the baculovirus expression vector pBlueBacHis2 B (Invitrogen, San Diego,Calif.) was selected as a source of control elements. The hTRT-codinginsert consisted of nucleotides 707 to 4776 of the hTRT insert inplasmid pGRN121.

A full length hTRT with a His6 and Anti-Xpress tags (Invitrogen) wasalso constructed. This vector also contains an insert consisting ofnucleotides 707 to 4776 of the hTRT insert from the plasmid pGRN121. Thevector directs expression in insect cells of high levels of full lengthhTRT protein fused to a cleavable 6-histidine and Anti-Xpress tags, andthe amino acid sequence of the fusion protein is shown below; (-*-)denotes enterokinase cleavage site:

MPRGSHHHHHHGMASMTGGQQMGRDLYDDDDL-*-DPSSRSAAGTMEFAAASTQRCVLLRTWEALAPATPAMPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHPRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD(SEQ ID NO:612)

Baculovirus Expression Vector pBlueBac4.5 and Full Length hTRT Protein

To produce large quantities of full-length, biologically active hTRT, asecond baculovirus expression vector, pBlueBac4.5 (Invitrogen, SanDiego, Calif.) was constructed. The hTRT-coding insert also consisted ofnucleotides 707 to 4776 of the hTRT from the plasmid pGRN121.

Baculovirus Expression Vector pMelBacB and Full Length hTRT Protein

To produce large quantities of full-length, biologically active hTRT, athird baculovirus expression vector, pMelBacB (Invitrogen, San Diego,Calif.) was constructed. The hTRT-coding insert also consists ofnucleotides 707 to 4776 of the hTRT insert from the plasmid pGRN121.

pMelBacB directs expression of full length hTRT in insect cells to theextracellular medium through the secretory pathway using the melittinsignal sequence. High levels of full length hTRT are thus secreted. Themelittin signal sequence is cleaved upon excretion, but is part of theprotein pool that remains intracellularly. For that reason, it isindicated in parentheses in the following sequence. The sequence of thefusion protein encoded by the vector is shown below:

(MKFLVNVALVFMVVYISYIYA)-*-DPSSRSAAGTMEFAAASTQRCVLLRTWEALAPATPAMPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFFPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD(SEQ ID NO:613)

Expression of hTRT in Mammalian Cells

The present invention also provides vectors to produce hTRT in largequantities as full-length, biologically active protein in a variety ofmammalian cell lines, which is useful in many embodiments of theinvention, as discussed above.

MPSV-hTRT Expression Plasmids

The invention also provides for an expression system for use inmammalian cells that gives the highest possible expression ofrecombinant protein, such as telomerase, without actually modifying thecoding sequence (e.g. optimizing codon usage). In one embodiment, theinvention provides MPSV mammalian expression plasmids (from plasmidpBBS212, described as pMPSV-TM in Lin J-H (1994) Gene 47:287-292)capable of expressing the TRTs of the invention. The MPSV plasmids canbe expressed either as stable or transient clones.

In this expression system, while the hTRT coding sequence itself isunchanged, exogenous transcriptional control elements are incorporatedinto the vector. The myeloproliferative sarcoma virus (MPSV) LTR(MPSV-LTR) promoter, enhanced by the cytomegalovirus (CMV) enhancer, isincorporated for transcriptional initiation. This promoter consistentlyshows higher expression levels in cell lines (see Lin J-H (1994) supra).A Kozak consensus sequence can be incorporated for translationinitiation (see Kozak (1996) Mamm. Genome 7:563-574). All extraneous 5′and 3′ untranslated hTRT sequences can be removed to insure that thesesequences do not interfere with expression, as discussed above. The MPSVplasmid containing the complete hTRT coding sequence, with allextraneous sequences included, is designated pGRN133. A control, hTRT“antisense” plasmid was also constructed. This vector is identical topGRN133 except that the TRT insert is the antisense sequence of hTRT(the antisense, which control can be used as a vector is designatedpGRN134). The MPSV plasmid containing the complete hTRT coding sequencewith all other extraneous sequences removed and containing the Kozakconsensus sequence is designated pGRN145.

Two selection markers, PAC (Puromycin-N-acetyl-transferase=Puromycinresistance) and HygB (Hygromycin B=Hygromycin resistance) are presentfor selection of the plasmids after transfection (see discussionreferring to selectable markers, above). Double selection using markerson both sides of the vector polylinker should increase the stability ofthe hTRT coding sequence. A DHFR (dihydrofolate reductase) encodingsequence is included to allow amplification of the expression cassetteafter stable clones are made. Other means of gene amplification can alsobe used to increase recombinant protein yields.

The invention also provides for MPSV mammalian expression plasmidscontaining hTRT fusion proteins. In one embodiment, the hTRT sequence,while retaining its 5′ untranslated region, is linked to an epitopeflag, such as the IBI FLAG (International Biotechnologies Inc. (IBI),Kodak, New Haven, Conn.) and inserted into the MPSV expression plasmid(designated pGRN147). This particular constuct contains a Kozaktranslation initiation site. The expressed fusion protein can bepurified using the M−1 anti-FLAG octapeptide monoclonal antibody (IBI,Kodak, supra).

In another embodiment, hTRT is site-specifically altered. One amino acidresidue codon is mutagenized, changing the aspartic acid at position 869to an alanine. This Asp869->Ala hTRT mutant, retaining its 5′untranslated region and incorporating a Kozak sequence, was insertedinto an MPSV expression plasmid, and designated pGRN146. The Asp869->AlahTRT mutant was further engineered to contain the FLAG sequence, asdescribed above, and the insert cloned into an MPSV expression plasmid.One such expression plasmid is designated pGRN154-I. Specifically, forpGRN154-I, an Eam1105I restriction digest fragment from pGRN146containing the Kozak sequence-containing “front end” (5′ segment) ofhTRT is cloned into the Eam1105I sites of pGRN147 (see above) to make anMPSV expression plasmid capable of expressing hTRT with a Kozaksequence, the above-described D869->A mutation, and the IBI flag.

Another embodiment of the invention is an expression plasmid derivedfrom pGRN146. The mammalian expression plasmid, designated pGRN152, wasgenerated by excising the EcoRI fragment from plasmid pGRN146(containing the hTRT ORF) and cloned into the EcoRI site of pBBS212 toremove the 5′UTR of hTRT. The hTRT is oriented so that its expression iscontrolled by the MPSV promoter. This makes a mammalian expressionplasmid that expresses hTRT with a Kozak consensus sequence and theD869->A mutation, and uses the MPSV promoter.

The invention provides for a mammalian expression vector in which hTRTis oriented so that the hTRT coding sequence is driven by the MPSVpromoter. For example, an EcoR1 restriction digest fragment from pGRN137containing the hTRT open reading frame (ORF) was cloned into the EcoR1site of pBBS212 (see below), thus removing the 5′ untranslated region(5′-UTR) of hTRT. pGRN137 was constructed by excising a SalI-Sse8387Ifragment from pGRN130, described below, containing the Kozak mutation ofhTRT into the Sal 1-SSE 8387I sites of pGRN136, making a mammalianexpression plasmid expressing hTRT containing a Kozak consensus sequenceoff the MPSV promoter. Plasmid pGRN136 was constructed by excising aHindIII SalI fragment from pGRN126 containing the hTRT ORF and cloningit into the HindIII SalI sites of plasmid, pBBS242, making a mammalianexpression plasmid expressing hTRT off the MPSV promoter). This makes amammalian expression plasmid, designated pGRN145, that expresses hTRTwith a Kozak consensus sequence using the MPSV promoter. See also thepGRN152 MPSV promoter-driven mammalian expression vector describedbelow.

hTRT Expressed in 293 Cells using Episomal Vector pEBVHis

An episomal vector, pEBVHis (Invitrogen, San Diego, Calif.) wasengineered to express an hTRT fusion protein comprising hTRT fused to anN-terminal extension epitope tag, the Xpress epitope (Invitrogen, SanDiego, Calif.) (designated pGRN122). The NotI hTRT fragment from pGRN121containing the hTRT ORF was cloned into the NotI site of pEBVHisA sothat the hTRT ORF is in the same orientation as the vector's RousSarcoma Virus (RSV) promoter. In this orientation the His6 flag wasrelatively closer to the N-terminus of hTRT.

A vector was also constructed containing as an insert the antisensesequence of hTRT and the epitope tag (the plasmid designated pGRN123,which can be used as a control). The vector was transfected into 293cells and translated hTRT identified and isolated using an antibodyspecific for the Xpress epitope. pEBVHis is a hygromycin resistant EBVepisomal vector that expresses the protein of interest fused to aN-terminal peptide. Cells carrying the vector are selected and expanded,then nuclear and cytoplasmic extracts prepared. These and controlextracts are immunoprecipitated with anti-Xpress antibody, and theimmunoprecipitated beads are tested for telomerase activity byconventional assay.

Expression of Recombinant hTRT in Mortal, Normal Diploid Human Cells

In one embodiment of the invention, recombinant hTRT and necessarytelomerase enzyme complex components can be expressed in normal, diploidmortal cells to increase their proliferative capacity or to immortalizethem, or to facilitate immortalizing them. This allows one to obtaindiploid immortal cells with an otherwise normal phenotype and karotype.As discussed above, this use of telomerase has enormous commercialutility.

Sense hTRT (FIG. 16) and antisense hTRT were cloned into a CMV vector.These vectors were purified and transiently transfected into two normal,mortal, diploid human cell clones. The human clones were young passagediploid human BJ and IMR90 cell strains.

Analysis of telomerase activity using a TRAP assay utilizing theTRAPeze™ Kit (Oncor, Inc., Gaithersburg, Md.) showed that transfectionof sense hTRT—but not antisense hTRT—generated telomerase activity inboth the BJ and IMR90 cell strains.

Expression of Recombinant hTRT in Immoralized IMR90 Human Cells

Using the same hTRT sense construct cloned into CMV vectors used in theabove described diploid human BJ and IMR90 cell strains studies,immortalized SW13 ALT pathway cell line (an IMR90 cell immortalized withSV40 antigen) was transiently transfected. A TRAP assay (TRAPeze, Oncor,Inc, Gaithersburg, Md.) demonstrated that telomerase activity wasgenerated in the sense construct transfected cells.

Vectors for Regulated Expression of hTRT in Mammalian Cells: Inducibleand Repressible Expression of hTRT

The invention provides vectors that can be manipulated to induce orrepress the expression of the TRTs of the invention, such as hTRT. Forexample, the hTRT coding sequence can be cloned into theEcdysone-Inducible Expression System from Invitrogen (San Diego, Calif.)and the Tet-On and Tet-off tetracycline regulated systems from ClontechLaboratories, Inc. (Palo Alto, Calif.). Such inducible expressionsystems are provided for use in the methods of the invention where it isimportant to control the level or rate of transcription of transfectedTRT. For example, the invention provides for cell lines immortalizedthrough the expression of hTRT; such cells can be rendered “mortal” byinhibition of hTRT expression by the vector through transcriptionalcontrols, such as those provided by the Tet-Off system. The inventionalso provides for methods of expressing TRT only transiently to avoidthe constitutive expression of hTRT, which may lead to unwanted“immortalization” of the transfected cells, as discussed above.

The Ecdysone-Inducible Mammalian Expression System is designed to allowregulated expression of the gene of interest in mammalian cells. Thesystem is distinguished by its tightly regulated mechanism that allowsalmost no detectable basal expression and greater than 200-foldinducibility in mammalian cells. The expression system is based on theheterodimeric ecdysone receptor of Drosophila. The Ecdysone-InducibleExpression System uses a steroid hormone ecdysone analog, muristerone A,to activate expression of hTRT via a heterodimeric nuclear receptor.Expression levels have been reported to exceed 200-fold over basallevels with no effect on mammalian cell physiology “Ecdysone-InducibleGene Expression in Mammalian Cells and Transgenic Mice” (1996) Proc.Natl. Acad. Sci. USA 93, 3346-3351). Once the receptor binds ecdysone ormuristerone, an analog of ecdysone, the receptor activates anecdysone-responsive promoter to give controlled expression of the geneof interest. In the Ecdysone-Inducible Mammalian Expression System, bothmonomers of the heterodimeric receptor are constitutively expressed fromthe same vector, pVgRXR. The ecdysone-responsive promoter, whichultimately drives expression of the gene of interest, is located on asecond vector, pIND, which drives the transcription of the gene ofinterest.

The hTRT coding sequence is cloned in the pIND vector (ClontechLaboratories, Inc, Palo Alto, Calif.), which contains 5 modifiedecdysone response elements (E/GREs) upstream of a minimal heat shockpromoter and the multiple cloning site. The construct is thentransfected in cell lines which have been pre-engineered to stablyexpress the ecdysone receptor. After transfection, cells are treatedwith muristerone A to induce intracellular expression from pIND.

The Tet-on and Tet-off expression systems (Clontech, Palo Alto, Calif.)give access to the regulated, high-level gene expression systemsdescribed by Gossen (1992) “Tight control of gene expression inmammalian cells by tetracycline responsive promoters” Proc. Natl. Acad.Sci. USA 89:5547-5551, for the Tet-Off transcription repression system;and Gossen (1995) “Transcriptional activation by tetracycline inmammalian cells” Science 268:1766-1769, for the Tet-On inducibletranscriptional system. In “Tet-Off” transformed cell lines, geneexpression is turned on when tetracycline (Tc) or doxycycline (“Dox;” aTc derivative) is removed from the culture medium. In contrast,expression is turned on in Tet-On cell lines by the addition of Tc orDox to the medium. Both systems permit expression of cloned genes to beregulated closely in response to varying concentrations of Tc or Dox.

This system uses the “pTRE” as a response plasmid that can be used toexpress a gene of interest. Plasmid pTRE contains a multiple cloningsite (MCS) immediately downstream of the Tet-responsive PhCMV*−1promoter. Genes or cDNAs of interest inserted into one of the sites inthe MCS will be responsive to the tTA and rtTA regulatory proteins inthe Tet-Off and Tet-On systems, respectively. PhCMV*−1 contains theTet-responsive element (TRE), which consists of seven copies of the42-bp tet operator sequence (tetO). The TRE element is just upstream ofthe minimal CMV promoter (PminCMV), which lacks the enhancer that ispart of the complete CMV promoter in the pTet plasmids. Consequently,PhCMV*−1 is silent in the absence of binding of regulatory proteins tothe tetO sequences. The cloned insert must have an initiation codon. Insome cases, addition of a Kozak consensus ribosome binding site mayimprove expression levels; however, many cDNAs have been efficientlyexpressed in Tet systems without the addition of a Kozak sequence.pTRE-Gene X plasmids are cotransfected with pTK-Hyg to permit selectionof stable transfectants.

Setting up a Tet-Off or Tet-On expression system generally requires twoconsecutive stable transfections to create a “double-stable” cell linethat contains integrated copies of genes encoding the appropriateregulatory protein and TRT under the control of a TRE. In the firsttransfection, the appropriate regulatory protein is introduced into thecell line of choice by transfection of a “regulator plasmid” such aspTet-Off or pTet-On vector, which expresses the appropriate regulatoryproteins. The hTRT cloned in the pTRE “response plasmid” is thenintroduced in the second transfection to create the double-stableTet-Off or Tet-On cell line. Both systems give very tight on/off controlof gene expression, regulated dose-dependent induction, and highabsolute levels of gene expression.

Expression Recombinant hTRT With DHFR and Adenovirus Sequences

The pGRN155 plasmid construct was designed for transient expression ofhTRT cDNA in mammalian cells. A Kozak consensus is inserted at the 5′end of the hTRT sequence. The hTRT insert contains no 3′ or 5′ UTR. ThehTRT cDNA is inserted into the EcoRI site of p91023(B) (Wong (1985)Science 228:810-815). The hTRT insert is in the same orientation as theDHFR ORF.

Plasmid pGRN155 contains the SV40 origin and enhancer just upstream ofan adenovirus promoter, a tetracycline resistance gene, an E. coliorigin and an adenovirus VAI and VAII gene region. This expressioncassette contains, in the following order: the adenovirus major latepromoter; the adenovirus tripartite leader; a hybrid intron consistingof a 5′ splice site from the first exon of the tripartite leader and a3′ splice site from the mouse immunoglobulin gene; the hTRT cDNA; themouse DHFR coding sequence; and, the SV40 polyadenylation signal.

The adenovirus tripartite leader and the VA RNAs have been reported toincrease the efficiency with which polycistronic mRNAs are translated.DHFR sequences have been reported to enhance the stability of hybridmRNA. DHFR sequences also can provide a marker for selection andamplification of vector sequences. See Logan (1984) Proc. Natl Acad.Sci. USA 81:3655); Kaufman (1985) Proc. Natl. Acad. Sci. USA 82: 689;and Kaufman (1988) Focus (Life Technologies, Inc.), Vol.10, no. 3). Thismakes the expression vector particularly useful for transientexpression.

Other expression plamids of the invention are described for illustrativepurposes.

pGRN121

The EcoRI fragment from lambda clone 25-1.1.6 containing the entire cDNAencoding hTRT protein was inserted into the EcoRI site ofpBluescriptIISK+such that the 5′ end of the cDNA is near the T7 promoterin the vector. The selectable marker that is used with this vector isampicillin.

pGRN122

The NotI fragment from pGRN121 containing the hTRT ORF was inserted intothe NotI site of pEBVHisA so that the coding sequence is operably linkedto the RSV promoter. This plasmid expresses a fusion protein composed ofa His6 flag fused to the N-terminal of the hTRT protein. The selectablemarker that is used with this vector is ampicillin or hygromycin.

pGRN123

The NotI fragment from pGRN121 containing the hTRT ORF was inserted intothe NotI site of pEBVHisA so that the coding sequence is in the oppositeorientation as the RSV promoter, thus expressing antisense hTRT.

pGRN124

Plasmid pGRN121 was deleted of all ApaI sites followed by deletion ofthe MscI-HincII fragment containing the 3′UTR. The Nco-XbaI fragmentcontaining the stop codon of the hTRT coding sequence was then insertedinto the Nco-XbaI sites of pGRN121 to make a plasmid equivalent topGRN121 except lacking the 3′UTR, which may be preferred for increasedexpression levels in some cells.

pGRN125

The NotI fragment from pGRN124 containing the hTRT coding sequence wasinserted into the NotI site of pBBS235 so that the open reading frame isin the opposite orientation of the Lac promoter. The selectable markerthat is used with this vector is chloramphenicol.

pGRN126

The NotI fragment from pGRN124 containing the hTRT coding sequence wasinserted into the NotI site of pBBS235 so that the hTRT coding sequenceinserted is in the same orientation as the Lac promoter.

pGRN127

The oligonucleotide5′-TGCGCACGTGGGAAGCCCTGGCagatctgAattCcaCcATGCCGCGCGCTCCCCGCTG-3′ (SEQ IDNO:608) was used in in vitro mutagenesis of pGRN125 to convert theinitiating ATG codon of the hTRT coding sequence into a Kozak consensussequence and create EcoRI and BglII sites for cloning. Also,oligonucleotide COD2866 was used to convert AmpS to AmpR (ampicillinresistant) and oligonucleotide COD1941 was used to convert CatR(chloramphenicol resistant) to CatS (chloramphenicol sensitive).

pGRN128

The oligonucleotide5′-TGCGCACGTGGGAAGCCCTGGCagatctgAattCcaCcATGCCGCGCGCTCCCCGCTG-3′ (SEQ IDNO:608) is used in in vitro mutagenesis to convert the initiating ATGcodon of hTRT into a Kozak consensus and create EcoRI and BglII sitesfor cloning. Also, oligo5′-CTGCCCTCAGACTTCAAGACCATCCTGGACTACAAGGACGACGATGACAAATGAATTCAGATCTGCGGCCGCCACCGCGGTGGAGCTCCAGC-3′(SEQ ID NO:614) is used to insert the IBI Flag (InternationalBiotechnologies Inc. (131), Kodak, New Haven, Conn.) at the C-terminusand create EcoRI and BglII sites for cloning. Also, COD2866 is used toconvert AmpS to AmpR and COD1941 is used to convert CatR to CatS.

pGRN129

The oligonucleotide5′-CGGGACGGGCTGCTCCTGCGTTTGGTGGAcGcgTTCTTGTTGGTGACACCTCACCTCACC-3′ (SEQID NO:609) was used by in vitro mutagenesis to convert Asp869 to an Alacodon (i.e. the second Asp of the DD motif was converted to an Alanineto create a dominant/negative hTRT mutant). This also created a MluIsite. Also, oligonucleotide5′-CTGCCCTCAGACTTCAAGACCATCCTGGACTACAAGGACGACGATGACAAATGAATTCAGATCTGCGGCCGCCACCGCGGTGGAGCTCCAGC-3′SEQ ID NO:614) was used to insert the IBI Flag at the C-terminus andcreate EcoRI and BglII sites for cloning. Also, COD2866 was used toconvert AmpS to AmpR and COD1941 was used to convert CatR to CatS.

pGRN130

The oligonucleotide5′-CGGGACGGGCTGCTCCTGCGTTTGGTGGAcGcgTTCTTGTTGGTGACACCTCACCTCACC-3′ (SEQID NO:609) was used in in vitro mutagenesis to convert the Asp869 codoninto an Ala codon (i.e. the second Asp of the DD motif was converted toan Alanine to make a dominant/negative variant protein). This alsocreated an MluI site. Also, the oligonucleotide5′-TGCGCACGTGGGAAGCCCTGGCagatctgAattCcaCcATGCCGCGCGCTCCCCGCTG-3′ (SEQ IDNO:608) was used in in vitro mutagenesis to convert the initiating ATGcodon of the hTRT coding sequence into a Kozak consensus sequence andcreate EcoRI and BglII sites for cloning. Also, COD2866 was used toconvert AmpS to AmpR and COD1941 was used to convert CatR.

pGRN131

The EcoRI fragment from pGRN128 containing the hTRT ORF with Kozaksequence and IBI Flag mutations is inserted into the EcoRI site ofpBBS212 so that the hTRT ORF is expressed off the MPSV promoter. PlasmidpBSS212 contains a MPSV promoter, the CMV enhancer, and the SV40polyadenylation site.

pGRN132

The EcoRI fragment from pGRN128 containing the hTRT ORF with Kozaksequence and 1131 Flag mutations is inserted into the EcoRI site ofpBBS212 so that the antisense of the hTRT ORF is expressed off the MPSVpromoter.

pGRN133

The EcoRI fragment from pGRN121 containing the hTRT coding sequence wasinserted into the EcoRI site of pBBS212 so that the hTRT protein isexpressed under the control of the MPSV promoter.

pGRN134

The EcoRI fragment from pGRN121 containing the hTRT coding sequence wasinserted into the EcoRI site of pBBS212 so that the antisense of thehTRT coding sequence is expressed under the control of the MPSVpromoter. The selectable markers used with this vector areChlor/HygB/PAC.

pGRN135

Plasmid pGRN126 was digested to completion with MscI and SmaI andreligated to delete over 95% of the hTRT coding sequence inserted. OneSmaI-MscI fragment was re-inserted during the process to recreate theCat activity for selection. This unpurified plasmid was then redigestedwith SalI and EcoRI and the fragment containing the initiating codon ofthe hTRT coding sequence was inserted into the SalI-EcoRI sites ofpBBS212. This makes an antisense expression plasmid expressing theantisense of the 5′UTR and 73 bases of the coding sequence. Theselectable markers used with this vector are Chlor/HygB/PAC.

pGRN136

The HindIII-SalI fragment from pGRN126 containing the hTRT codingsequence was inserted into the HindIII-SalI sites of pBBS242.

pGRN137

The SalI-Sse8387I fragment from pGRN130 containing the Kozak sequencewas inserted into the SalI-Sse8387I sites of pGRN136.

pGRN138

The EcoRI fragment from pGRN124 containing hTRT minus the 3′UTR wasinserted into the EcoRI site of pEGFP-C2 such that the orientation ofthe hTRT is the same as the EGFP domain.

pGRN139

The oligonucleotide5′-CTGCCCTCAGACTTCAAGACCATCCTGGACTACAAGGACGACGATGACAAATGAATTCAGATCTGCGGCCGCCACCGCGGTGGAGCTCCAGC-3′(SEQ ID NO:614) was used to insert the IBI Flag at the C-terminus ofhTRT in pGRN125 and create EcoRI and BglII sites for cloning. Also,COD2866 was used to convert AmpS to AmpR and COD 1941 was used toconvert CatR to CatS.

pGRN140

The NcoI fragment containing the upstream sequences of genomic hTRT andthe first intron of hTRT from lambdaG55 was inserted into the NcoI siteof pBBS167. The fragment is oriented so that hTRT is in the samedirection as the Lac promoter.

pGRN141

The NcoI fragment containing the upstream sequences of genomic hTRT andthe first intron of hTRT from lambdaG55 was inserted into the NcoI siteof pBBS167. The fragment is oriented so that hTRT is in the oppositedirection as the Lac promoter.

pGRN142

The NotI fragment from lambdaGphi5 containing the complete˜15 kbpgenomic insert including the hTRT gene promoter region was inserted inthe NotI site of plasmid pBBS 185. The fragment is oriented so that thehTRT ORF is in the opposite orientation as the Lac promoter.

pGRN143

The NotI fragment from lambdaGphi5 containing the complete˜15 kbpgenomic insert including the hTRT gene promoter region was inserted inthe NotI site of plasmid pBBS 185. The fragment is oriented so that thehTRT ORF is in the same orientation as the Lac promoter.

pGRN144

SAL1 deletion of pGRN140 to remove lambda sequences.

pGRN145

This vector was constructed for the expression of hTRT sequences inmammalian cells. The EcoRI fragment from pGRN137 containing the hTRTcoding sequence was inserted into the EcoRI site of pBBS212 to removethe portion of the sequence corresponding to the 5′UTR of hTRT mRNA. ThehTRT coding sequence is oriented so that it is expressed under thecontrol of the MPSV promoter. The selectable markers used with thisvector are Chlor/HygB/PAC.

pGRN146

This vector was constructed for the expression of hTRT sequences inmammalian cells. The Sse8387I-NotI fragment from pGRN130 containing theD869A mutation of hTRT was inserted into the Sse8387I-NotI sites ofpGRN137. The selectable markers used with this vector areAmpicillin/HygB/PAC.

pGRN147

The Sse8387I-NotI fragment from pGRN139 containing the IBI Flag wasinserted into the Sse8387I-NotI sites of pGRN137.

pGRN148

The BglII-Eco47III fragment from pGRN144 containing the promoter regionof hTRT was inserted into the BglII-NruI sites of pSEAP2 to make an hTRTpromoter/reporter construct.

pGRN149

This vector is an intermediate vector for constructing a hTRT fusionprotein expression vector. The mutagenic oligo5′-cttcaagaccatcctggactttcgaaacgcggccgccaccgcggtggagctcc-3′ (SEQ IDNO:615) was used to add a CSP45I site at the C-terminus of hTRT by invitro mutagenesis of pGRN125. The “stop” codon of hTRT was deleted andreplaced with a Csp45I site. The selectable marker that is used withthis vector is ampicillin.

pGRN150

The BglII-FspI fragment from pGRN144 containing the promoter region ofhTRT was inserted into the BglII-NruI sites of pSEAP2 to make an hTRTpromoter/reporter construct.

pGRN151

This vector was constructed for the expression of hTRT sequences inmammalian cells. The EcoRI fragment from pGRN147 containing the hTRTcoding sequence was inserted into the EcoRI site of pBBS212 to removethe portion of the sequence corresponding to the 5′UTR of the hTRT mRNA.The hTRT coding sequence is oriented so that it is expressed under thecontrol of the MPSV promoter. The selectable markers used with thisvector are Chlor/HygB/PAC.

pGRN152

The EcoRI fragment from pGRN146 containing the hTRT coding sequence wasinserted into the EcoRI site of pBBS212 to remove the portion of thesequence corresponding to the 5′UTR of the hTRT. The hTRT codingsequence is oriented so that it is expressed under the control of theMPSV promoter.

pGRN153

The StyI fragment from pGRN130 containing the D869→A mutation of hTRT(hTRT variant coding sequence) was inserted into the StyI sites ofpGRN158 to make a plasmid containing the hTRT coding sequence with aKozak consensus sequence at its 5′-end, an IBI FLAG sequence at its3′-end (the C-terminus encoding region), and the D869-->A mutation.

pGRN154

The EcoRI fragment of pGRN153 containing the hTRT gene was inserted intothe EcoRI site of plasmid pBS212 in an orientation such that the hTRTORF is oriented in the same direction as the MPSV promoter. This makesan MPSV-directed expression plasmid that expresses the hTRT protein witha Kozak consensus sequence at its amino-terminal end, an IBI FLAG at itscarboxy-terminal end, and the D869→A mutation

pGRN155

This vector was constructed for the expression of hTRT sequences inmammalian cells. The insert included full length cDNA of hTRT minus 5′and 3′ UTR, and Kozak sequences. The EcoRI fragment from pGRN145containing the hTRT cDNA with the Kozak consensus and no 3′ or 5′ UTRwas inserted into the EcoRI site of p91023(B) such that the hTRT is inthe same orientation as the DHFR ORF. This makes a transient expressionvector for hTRT. The selectable marker used with this vector istetracycline.

pGRN156

This vector was constructed for the expression of hTRT sequences inmammalian cells. The EcoRI fragment from pGRN 146 containing the D869Amutation of the hTRT cDNA with the Kozak consensus and no 3′ or 5′ UTRwas inserted into the EcoRI site of p91023(B) such that the hTRT is inthe same orientation as the DHFR ORF. This makes a transient expressionvector for hTRT. The insert included full length cDNA of hTRT minus 5′and 3′ UTR, D869A, and Kozak sequences. The selectable marker used withthis vector is tetracycline.

pGRN157

This vector was constructed for the expression of hTRT sequences inmammalian cells. The EcoRI fragment from pGRN147 containing the hTRTcDNA with the IBI FLAG at the C-terminus; the Kozak consensus and no 3′or 5′ UTR into the EcoRI site of p91023(B) such that the hTRT is in thesame orientation as the DHFR ORF. This makes a transient expressionvector for hTRT. The insert included full length cDNA of hTRT minus 5′and 3′ UTR, the IBI FLAG sequence, and Kozak sequences. The selectablemarker used with this vector is tetracycline.

pGRN158

This vector was constructed for the expression and mutagenesis of TRTsequences in E. coli. The EcoRI fragment from pGRN151 containing thehTRT ORF was inserted into the EcoRI site of pBBS183 so that the hTRTORF is oriented in the opposite direction as the Lac promoter. Theinsert included full length cDNA of hTRT minus 5′ and 3′ UTR, IBI FLAGsequence, and Kozak sequences. The hTRT coding sequence is driven by aT7 promoter. The selectable marker used with this vector is amphicillin.

pGRN159

This vector was constructed for the expression and mutagenesis of TRTsequences in E. coli. The NheI-KpnI fragment from pGRN138 containing theEGFP to hTRT fusion was inserted into the XbaI-KpnI sites ofpBluescriptIIKS+. This makes a T7 expression vector for the fusionprotein (the coding sequence is driven by a T7 promoter). The insertincluded full length cDNA of hTRT minus the 3′ UTR as a fusion proteinwith EGFP. The selectable marker used with this vector is amphicillin.

pGRN160

This vector was constructed for the expression of antisense hTRsequences in mammalian cells. The coding sequence is operably linked toan MPSV promoter. The XhoI-NsiI fragment from pGRN90 containing the fulllength hTR ORF was inserted into the SalI-Sse8387I sites of pBBS295.This makes a transient/stable vector expressing hTR antisense RNA. A GPTmarker was incorporated into the vector. The selectable markers usedwith this vector are Chlor/gpt/PAC.

pGRN161

This vector was constructed for the expression of sense hTR sequences inmammalian cells. The XhoI-NniI fragment from pGRN89 containing the fulllength hTR ORF was inserted into the SalI-Sse8387I sites of pBBS295.This makes a transient/stable vector expressing hTR in the senseorientation. The coding sequence is driven by an MPSV promoter. A GPTmarker was incorporated into the vector. The selectable markers usedwith this vector are Chlor/gpt/PAC.

pGRN162

The XhoI-NsiI fragment from pGRN87 containing the full length hTR ORFwas inserted into the SalI-Sse8387I sites of pBBS295. This makes atransient/stable vector expressing truncated hTR (from position +108 to+435) in the sense orientation.

pGRN163

This vector was constructed for the expression and mutagenesis of TRTsequences in E. coli. The coding sequence is driven by a T7 promoter.Oligonucleotide RA45 (5′-GCCACCCCCGCGCTGCCTCGAGCTCCCCGCTGC-3′; SEQ IDNO:616) is used in in vitro mutagenesis to change the initiating met inhTRT to Leu and introduce an XhoI site in the next two codons after theLeu. Also COD 1941 was used to change CatR to CatS, and introduces aBSPH1 site, and COD 2866 was used to change AmpS to AmpR, introducing anFSP1 site. The selectable marker used with this vector is amphicillin.

pGRN164

This vector was constructed for the expression of hTR sequences in E.coli. Primers hTR+1 5′-GGGGAAGCTTTAATACGACTCACTATAGGGTTGCGGAGGGTGGGCCTG-3′ (SEQ ID NO:617) and hTR+4455′-CCCCGGATCCTGCGCATGTGTGAGCCGAGTCCTGGG-3′ (SEQ ID NO:618) were used toamplify by PCR a fragment from pGRN33 containing the full length hTRwith the T7 promoter on the 5′ end (as in hTR+1). A BamHI-HindIII digestof the PCR product was put into the BamHI-HindIII sites of pUC119. Thecoding sequence operably linked to a T7 promoter. The selectable markerused with this vector is amphicillin. pGRN164 is also called phTR+1.

pGRN165

This vector was constructed for the expression and mutagenesis of hTRTsequences in E. coli. The coding sequence is operably linked to a T7promoter. The EcoRI fragment from pGRN145 containing the hTRT ORF with aKozak front end was inserted into the EcoRI site of pBluescriptIISK+ sothat the hTRT is oriented in the same direction as the T7 promoter. Theselectable marker used with this vector is amphicillin.

pGRN166

This vector was constructed for the expression and mutagenesis of TRTsequences in mammalian cells. The coding sequence is operably linked toa T7 promoter. The EcoRI fragment from pGRN151 containing the hTRT ORFwith a Kozak front end and IBI flag at the back end was inserted intothe EcoRI site of pBluescriptIISK+ so that the hTRT ORF is oriented inthe same direction as the T7 promoter. The insert included full lengthcDNA of hTRT minus 5′ and 3′ UTR, FLAG sequence (Immunex Corp, SeattleWash.), and Kozak sequences. The selectable marker used with this vectoris amphicillin.

pGRN167

AvRII-StuI fragment from pGRN144 containing the 5′ end of the hTRT ORFwas inserted into the XbaI-StuI sites of pBBS161.

pGRN168

The EcoRI fragment from pGRN145 containing the optimized hTRT expressioncassette was inserted into the EcoRI site of pIND such that the hTRTcoding sequence is in the same orientation as the miniCMV promoter.

pGRN169

The EcoRI fragment from pGRN145 containing the optimized hTRT expressioncassette was inserted into the EcoRI site of pIND such that the hTRT isin the reverse orientation from the miniCMV promoter.

pGRN170

The EcoRI fragment from pGRN145 containing the optimized hTRT expressioncassette was inserted into the EcoRI site of pIND(sp1) such that thehTRT is in the opposite orientation from the miniCMV promoter.

pGRN171

The Eco47III-NarI fragment from pGRN163 was inserted into theEco47III-NarI sites of pGRN167, putting the M1L mutation into a fragmentof the hTRT genomic DNA.

pGRN172

The BamHI-StuI fragment from pGRN171 containing the Met to Leu mutationin the hTRT ORF was inserted into the BglII-NruI sites of pSEAP2-Basic.

pGRN173

The EcoRV-ECO47III fragment from pGRN144 containing the 5′ end of thehTRT promoter region was inserted into the SrfI-Eco47III sites ofpGRN172. This makes a promoter reporter plasmid that contains thepromoter region of hTRT from approximately 2.3 kb upstream from thestart of the hTRT ORF to just after the first intron in the codingregion, with the Met1→Leu mutation.

pGRN174

The EcoRI fragment from pGRN145 containing the “optimized” hTRTexpression cassette was inserted into the EcoRI site of pIND(sp1) suchthat the hTRT is in the same orientation as the miniCMV promoter.

Example 7 Reconstitution of Telomerase Activity

A. Co-Expression of hTRT and hTR in vitro

In this example, the coexpression of hTRT and hTR using an in vitrocell-free expression system is described. These results demonstrate thatthe hTRT polypeptide encoded by pGRN121 encodes a catalytically activetelomerase protein and that in vitro reconstitution (IVR) of thetelomerase RNP can be accomplished using recombinantly expressed hTRTand hTR.

Telomerase activity was reconstituted by adding linearized plasmids ofhTRT (pGRN121; 1 μg DNA digested with Xba I) and hTR (phTR+1; 1 μgdigested with FspI) to a coupled transcription-translation reticulocytelysate system (Promega TNT™). phTR+1 is a plasmid which, when linearizedwith FspI and then transcribed by T7 RNA polymerase, generates a 445nucleotide transcript beginning with nucleotide+1 and extending tonucleotide 446 of hTR (Autexier et al., 1996, EMBO J 15:5928). For a 50μl reaction the following components were added: 2 μl TNT™ buffer, 1 μlTNT™ T7 RNA polymerase, 1 μl 1 mM amino acid mixture, 40 units Rnasin™RNase inhibitor, 1 μg each linearized template DNA, and 25 μl TNT™reticulocyte lysate. Components were added in the ratio recommended bythe manufacturer and were incubated for 90 min at 30° C. Transcriptionwas under the direction of the T7 promoter and could also be carried outprior to the addition of reticulocyte lysate with similar results. Afterincubation, 5 and 10 μl of the programmed transcription-translationreaction were assayed for telomerase activity by TRAP as previouslydescribed (Autexier et al., supra) using 20 cycles of PCR to amplify thesignal.

The results of the reconstitution are shown in FIG. 10. For eachtranscription!translation reaction assayed there are 3 lanes: The first2 lanes are duplicate assays and the third lane is a duplicate sampleheat denatured (95° C., 5 min) prior to the TRAP phase to rule out PCRgenerated artifacts.

As shown in FIG. 10, reticulocyte lysate alone has no detectabletelomerase activity (lane 6). Similarly, no detectable activity isobserved when either hTR alone (lane 1) or full length hTRT gene (lane4) are added to the lysate. When both components are added (lane 2),telomerase activity is generated as demonstrated by the characteristicrepeat ladder pattern. When the carboxyl-terminal region of the hTRTgene is removed by digestion of the vector with NcoI (“truncated hTRT”)telomerase activity is abolished (lane 3). Lane 5 shows that translationof the truncated hTRT alone does not generate telomerase activity. Lane“R8” shows a positive control for a telomerase product ladder generatedby TRAP of TSR8, a synthetic telomerase product having a nucleotidesequence of 5′-ATTCCGTCGAGCAGAGTTAG[GGTTAG]₇-3′ (SEQ ID NO:619).

It was also observed that purification of IVR telomerase resulted in astronger signal and/or reduced background in certain telomerase activityassays. In some experiments, IVR telomerase activity from co-synthesizedcomponents was enriched by fractionation of TNT reactions over DEAEanion exchange membranes (Millipore Ultrafree-MC): 200 μl of thehTRT/hTR TNT reaction was passed through a single DEAE membrane. Themembrane was washed with 400 μl of 0.2 M NaCl in buffer A (20 mMHEPES-KOH pH 7.9, 2 mM MgCl₂, 1 mM EGTA, 10% glycerol, 0.1% NonidetP-40, 0.1 mM phenylmethylsulfonyl fluoride) and IVR telomerase waseluted from the membrane with 80 μl of 1 M NaCl in buffer A.Alternatively, batch chromatography was used: 400 μl of the TNT reactionwas partially purified by batch chromatography using 25 μl of Toso-HaasQ-650M resin. After binding telomerase to the resin, it was washed with0.1 M NaCl in buffer A, followed by a second wash with 0.18 M NaCl inbuffer A and eluted with 100 μl of 0.3 M NaCl in buffer A.

B. Mixing of hTRT and hTR in vitro

In vitro reconstitution of telomerase activity was also accomplished bymixing. hTRT was transcribed and translated as described supra, butwithout the addition of the hTR plasmid. Reconstitution of thetelomerase RNP was then accomplished by mixing the hTRT translationmixture with hTR (previously generated by T7 RNA polymerasetranscription from phTR+1-Fsp) in the ratio of 2 μl of hTRT translationmix to 2 μl of hTR (1 ug) then incubated for 90 minutes at 30° C. Thereaction conditions were adjusted to a KCl concentration of about 0.2 M.(The presence of KCl at a concentration of about 0.1 M to about 1.0 Mmay enhance telomerase activity or telomerase reconstitution in IVR).This method of hTRT/hTR reconstitution is referred to as “linkedreconstitution” or “linked IVR.” Telomerase activity is present (i.e.,can be detected) in this mixture. Improved signal was observed followingpartial purification of the activity by DEAE chromatography. In thiscase Millipore Ultrafree-MC DEAE Centrifugal Filter Devices were usedaccording to the manufacturer's directions). The buffers used werehypo0.1, hypo0.2, and hypo1.0, where hypo is 20 mM Hepes-KOH, pH 7.9, 2mM MgCl₂, 1 mM EGTA, 10% glycerol, 0.1% NP-40, 1 mM DTT, 1 mMNa-metabisulfite, 1 mM benzamidine, and 0.2 mMphenylmethylsulfonylflouride (PMSF), and where 0.1, 0.2 and 1.0 refersto 0.1, 0.2 or 1.0 M KCL. The filters were pre-conditioned with hypo1.0,washed with hypo0.1, the reconstituted telomerase was loaded, the columnwas washed with hypo0.1 then hypo0.2, and the reconstituted telomerasewas eluted with hypo1.0 at half the volume as was loaded. Thisformulation could be stored frozen at −70° C. and retains activity.

Telomerase activity was assayed in a two step procedure. In step one, aconventional telomerase assay was performed as described in Morin, 1989,Cell 59: 521, except no radiolabel was used. In step two, an aliquot wasassayed by the TRAP procedure for 20-30 cycles as described supra. Theconventional assay was performed by assaying 1-10 μl of reconstitutedtelomerase in 40-50 μl final volume of 25 mM Tris-HCl, pH 8.3, 50 mMK-acetate, 1 mM EGTA, 1 mM MgCl₂, 2 mM dATP, 2 mM TTP, 10 uM dGTP, and 1uM primer (usually M2, 5′-AATCCGTCGAGCAGAGTT; SEQ ID NO:620) at 30° C.for 60-180 minutes. The reaction was stopped by heating to 95° C. for 5minutes and 1-10 μl of the first step mixture was carried onto the steptwo TRAP reaction (50 ul).

In additional experiments, the synthesis of hTRT and hTR during in vitroreconstitution was monitored by ³⁵S-methionine incorporation andNorthern blotting, respectively. Proteins of approximately the predictedsize were synthesized for hTRT (127 kD), hTRT-Nco (85 kD), and pro90hTRT(90 kD) in approximately equal molar amounts relative to each other. TheNorthern analysis indicated hTR synthesis was the correct size (445nucleotides) and predominantly intact.

High levels of reconstitution and telomerase activity were also obtainedwith 2 μg of linearized pGRN121 in a 50 μl TnT reaction as describedsupra (Example 7A) except that in place of the hTR template, 4 pmol (0.6μg) of hTR RNA (previously generated by T7 RNA polymerase transcriptionfrom phTR+1-Fsp) was added at the beginning of the TnT reaction and thereaction was incubated at 30EC for 90-120 minutes. Slightly greater (2-5times) activity was achieved using 1 μg of supercoiled XhTRT- and 16pmol (2.4 μg) of pre-synthesized hTR RNA set up in a 50 μl TnT reaction,as described supra, with incubation at 30EC for 90-120 minutes. XhTRT-Eis an hTRT construct in the pcDNA3.1/His Xpress vector (Invitrogen) inwhich an optimized ribosome recognition site (Kozak consensus), sixhistidine residues, and an epitope tag are fused with the hTRT openreading frame.

Variations of the reconstitution protocols, supra, will be apparent tothose of skill. For example, the time and temperature of reconstitution,and presence or concentration of components such as monovalent salt(e.g. NaCl, KCl, potassium acetate, potassium glutamate, and the like),divalent salt (MgCl₂, MnCl₂, MgSO₄, and the like), denaturants (urea,formamide, and the like), detergents (NP-40, Tween, CHAPS, and thelike), and alternative improved purification procedures (such asimmunoprecipitation, affinity or standard chromatography) can beemployed. These and other parameters can be varied in a systematic wayto optimize conditions for particular assays or other reconstitutionprotocols.

C. Reconstitution Using hTRT Variants and Fusion Proteins

Reconstitution of telomerase catalytic activity occurred when EGFP-hTRT,a fusion of the enhanced green fluorescent protein to hTRT (see Examples6 and 15), or epitope-tagged hTRT (IBI FLAG, see Example 6) bothreconstituted telomerase activity at approximately wild-type levels werecoexpressed with hTR.

In contrast, telomerase activity was not reconstituted when a varianthTRT, pro90hTRT (missing RT motifs B′, C, D, and E) was used. Thisdemonstrates that pro90hTRT does not possess full telomerase catalyticactivity, although it may have other partial activities (e.g., RNA [i.e.hTR] binding ability and function as dominant-negative regulator oftelomerase in vivo as described supra).

D. Assay of in vitro Reconstituted Telomerase Activity Using the GelBlot and Conventional Telomerase Assay

The following example demonstrates that in vitro reconstituted (IVR)telomerase can be assayed using conventional telomerase assays inaddition to amplification-based assays (i.e., TRAP). IVR telomerase asdescribed in part (B), supra (the “linked reconstitution method”)followed by DEAE purification, as described supra was assayed using thegel blot assay using the following reaction conditions; 1-10 μl oflinked IVR telomerase in 40 μl final volume of 25 mM Tris-HCl, pH 8.3,50 mM K-acetate, 1 mM EGTA, 1 mM MgCl₂, 0.8 mM dATP, 0.8 mM TTP, 1.0 mMdGTP, and 1 uM primer ( M2, supra; or H3.03, 5′-TTAGGGTTAGGGTTAGGG; SEQID NO:621) at 30° C. for 180 minutes. The telomeric DNA synthesized wasisolated by standard procedures, separated on a 8% polyacrylamide, 8 Murea gel, transferred to a nylon membrane, and probed using the³²P-(CCCTAA)n riboprobe used in the dot-blot assay. The probe identifieda six nucleotide ladder in the lane representing 10 μl of IVR telomerasethat was equivalent to the ladder observed for 5 μl of native nucleartelomerase purified by mono Q and heparin chromatography. The resultsshow that IVR telomerase possesses processive telomerase catalyticactivity equivalent to native telomerase.

Linked IVR telomerase was also assayed by the conventional ³²P-dGTPincorporation telomerase assay. IVR telomerase prepared by the linkedreconstitution method followed by DEAE purification, as described above,was assayed under both processive and non-processive reactionconditions. Assay conditions were 5-10 μl of linked IVR telomerase in 40μl final volume of 25 mM Tris-HCl, pH 8.3, 50 mM K-acetate, 1 mM EGTA, 1mM MgCl₂, 2 mM dATP, 2 mM TTP, with 10 uM ³²P-dGTP (72 Ci/mmol) [forassay of processive conditions] or 1 uM ³²P-dGTP (720 Ci/mmol) [fornon-processive], and 1 uM primer (i.e., H3.03, supra) at 30° C. [for theprocessive reaction] or 37° C. [for the non-processive reaction] for 180minutes. The telomeric DNA synthesized was isolated by standardprocedures and separated on a 8% polyacrylamide, 8 M urea gel sequencinggel. The processive reaction showed a weak six nucleotide ladderconsistent with a processive telomerase reaction, and the non-processivereaction added one repeat, a pattern equivalent to a control reactionwith a native telomerase preparation. Conventional assays using IVRtelomerase are useful in screens for telomerase modulators, as describedherein, as well as other uses such as elucidation of the structural andfunctional properties of telomerase.

E. In vitro Reconstituted Telomerase Recognizes Primer 3′ Termini

This experiment demonstrates that IVR telomerase recognizes primer 3′termini equivalently to native (purified) telomerase. Telomerase forms abase-paired duplex between the primer 3′ end and the template region ofhTR and adds the next specified nucleotide (Morin, 1989, supra). Toverify that IVR (recombinant) telomerase has the same property, thereactions of primers with - - - GGG or - - - TAG 3′ termini(AATCCGTCGAGCAGAGGG; SEQ ID NO:622 and AATCCGTCGAGCAGATAG; SEQ IDNO:623) were compared to a primer having a - - - GTT 3′ terminus (M2supra) using IVR and native telomerase assayed by the two stepconventional/TRAP assay detailed above. The product ladders of the - - -GGG and - - - TAG primers were shifted +4 and +2, respectively, whencompared to the standard primer ( - - - GTT 3′ end), the same effect aswas observed with native telomerase. This experiment demonstrates IVRand native telomerases recognize primer termini in a similar manner.

These results (along with the results supra showing that IVR telomerasepossesses both processive and non-processive catalytic activity)indicate that IVR telomerase has similar structure and propertiescompared to native or purified telomerase.

Example 8 Production of Anti-hTRT Antibodies

A. Production of Anti-hTRT Antibodies Against hTRT Peptides

To produce anti-hTRT antibodies, the following peptides from hTRT weresynthesized with the addition of C (cysteine) as the amino terminalresidue (see FIG. 54).

S-1: FFY VTE TTF QKN RLF FYR KSV WSK SEQ ID NO:232

S-2: RQH LKR VQL RDV SEA EVR QHR EA SEQ ID NO:233

S-3: ART FRR EKR AER LTS RVK ALF SVL NYE SEQ ID NO:234

A-3: PAL LTS RLR FIP KPD GLR PIV NMD YVV SEQ ID NO:237

The cysteine moiety was used to immobilize (i.e., covalently link) thepeptides to BSA and KLH [keyhole limpet hemocyanin] carrier proteins.The KLH-peptides were used as antigen. The BSA-peptide conjugates servedas material for ELISAs for testing the specificity of immune antisera.

The KLH-peptide conjugates were injected into New Zealand White rabbits.The initial injections are made by placing the injectant proximal to theaxillary and inguinal lymph nodes. Subsequent injections were madeintramuscularly. For initial injections, the antigen was emulsified withFreund's complete adjuvant; for subsequent injections, Freund'sincomplete adjuvant was used. Rabbits follow a three week boost cycle,in which 50 ml of blood yielding 20-25 ml of serum is taken 10 daysafter each boost. Antisera against each of the four peptides recognizedthe hTRT moiety of recombinant hTRT fusion protein(GST-HIS₈-hTRT-fragment 2426 to 3274); see Example 6) on western blots.

Using a partially purified telomerase fraction from human 293 cells(approximately 1000-fold purification compared to a crude nuclearextract) that was produced as described in PCT application No. 97/06012and affinity purified anti-S-2 antibodies, a 130 kd protein doubletcould be detected on a western blot. A sensitive chemiluminescencedetection method was employed (SuperSignal chemiluminescence substrates,Pierce) but the signal on the blot was weak, suggesting that hTRT ispresent in low or very low abundance in these immortal cells. Theobservation of a doublet is consistent with a post-translationalmodification of hTRT, i.e., phosphorylation or glycosylation.

For affinity purification, the S-2 peptide was immobilized to SulfoLink(Pierce, Rockford Ill.) through its N-terminal Cysteine residueaccording to the manufacturer's protocol. First bleed serum from arabbit immunized with the KLH-S-2 peptide antigen was loaded over a theS-2-SulfoLink and antibodies specifically bound to the S-2 peptide wereeluted.

B. Production of Anti-hTRT Antibodies against hTRT Fusion Proteins

GST-hTRT fusion proteins were expressed in E. coli as the GST-hTRTfragment #4 (nucleotides 3272-4177) and the GST-HIS8-hTRT fragment #3(nucleotides 2426 to 3274) proteins described in Example 6. The fusionproteins were purified as insoluble protein, and the purity of theantigens was assayed by SDS polyacrylamide gels and estimated to beabout 75% pure for the GST-hTRT fragment #4 recombinant protein and morethan 75% pure for GST-HIS8-hTRT. fragment #3 recombinant protein.Routine methods may be used to obtain these and other fusion proteins ata purity of greater than 90%. These recombinant proteins were used toimmunize both rabbits and mice, as described above.

The first and second bleeds from both the mice and rabbits were testedfor the presence of anti-hTRT antibodies after removal of anti-GSTantibodies using a matrix containing immobilized GST. The antisera weretested for anti-hTRT antibodies by Western blotting using immobilizedrecombinant GST-hTRT fusion protein, and by immunoprecipitation usingpartially purified native telomerase enzyme. While no signal wasobserved in these early bleeds, titers of anti-hTRT antibodies, asexpected, increased in subsequent bleeds.

Example 9 Detection of an hTRT mRNA Corresponding to Δ182 RNA Variant

Poly A⁺ RNA from human testis and the 293 cell line was analyzed forhTRT mRNA using RT-PCR and nested primers. The first primer set wasTCP1.1 and TCP1.15; the second primer set was TCP1.14 and BTCP6.Amplification from each gave two products differing by 182 bp; thelarger and smaller products from testis RNA were sequenced and found tocorrespond exactly to pGRN121(FIG. 16) and the 712562 clone (FIG. 18),respectively. The variant hTRT RNA product has been observed in mRNAfrom SW39i, OVCAR4, 293, and Testes.

Additional experiments were carried out to demonstrate that the Δ182cDNA was not an artifact of reverse transcription. Briefly, full-lengthhTRT RNA (i.e., without the deletion) was produced by in vitrotranscription of pGRN121 for use as a template for RT-PCR. Separate cDNAsynthesis reactions were carried out using Superscript7 reversetranscriptase (Bethesda Research Laboratories, Bethesda Md.) at 42° or50° C., and with random-primers or a specific primer. After 15 PCRcycles the longer product was detectable; however, the smaller product(i.e., corresponding to the deletion) was not detectable even after 30or more cycles. This indicates that the RT-PCR product is not anartifact.

Example 10 Sequencing of Test is hTRT mRNA

The sequence of the testis form of hTRT RNA was determined by directmanual sequencing of DNA fragments generated by PCR from testis cDNA(Marathon Testes cDNA, Clontech, San Diego Calif.) using aThermoSequenase radiolabeled terminator cycle sequencing kit (AmershamLife Science). The PCR step was performed by a nested PCR, as shown inTable 8. In all cases a negative control reaction with primers but nocDNA was performed. The absence of product in the control reactiondemonstrated that the products derived from the reaction with cDNApresent were not due to contamination of hTRT from pGRN121 or other cellsources (e.g., 293 cells). The DNA fragments were excised from agarosegels to purify the DNA prior to sequencing.

The testis mRNA sequence corresponding to bases 27 to 3553 of thepGRN121 insert sequence, and containing the entire hTRT ORF (bases 56 to3451) was obtained. There were no differences between the testis and thepGRN121 sequences in this region.

TABLE 8 Final Fragment Primer Set 1 Primer Set 2 Size Primers for Seq OAna K320/K322 208 K320, K322 A K320/TCP1.43 TCP1.40/TCP1.34 556 TCP1.52,TCP1.39, K322, TCP1.40, TCP1.41, TCP1.30, TCP1.34, TCP1.49 BTCP1.42/TCP1.32B TCP1.35/TCP1.21 492 TCP1.35, TCP1.28, TCP1.38, TCP1.21,TCP1.46, TCP1.33, TCP1.48 C TCP1.65/TCP1.66 TCP1.67/TCP1.68 818 TCP1.67,TCP1.32, TCP1.69, TCP1.68, TCP1.24, TCP1.44, K303 D2 K304/billTCP6Lt1/TCP1.6 546 Lt2, Lt1, TCP1.6, billTCP4, TCP1.13, TCP1.77, TCP1.1 D3TCP1.12/TCP1.7 TCP1.14/TCP1.15 604 TCP1.6, TCP1.14, TCP1.73, TCP1.78,TCP1.25, TCP1.15, TCP1.76 EF na TCP1.74/TCP1.7 201 TCP1.74, TCP1.7,TCP1.75, TCP1.15, TCP1.3 E TCP1.3/TCP1.4 TCP1.2/TCP1.9 687 TCP1.2,TCP1.8, TCP1.9, TCP1.26 F TCP1.26/UTR2 TCP1.10 TCP1.4 377 TCP1.4,TCP1.10, TCP1.11

Example 11 Detection of hTRT mRNA by RNase Protection

RNase protection assays can be used to detect, monitor, or diagnose thepresence of an hTRT mRNA or variant mRNA. One illustrative RNAseprotection probe is an in vitro synthesized RNA comprised of sequencescomplementary to hTRT mRNA sequences and additional, non-complementarysequences. The latter sequences are included to distinguish thefull-length probe from the fragment of the probe that results from apositive result in the assay: in a positive assay, the complementarysequences of the probe are protected from RNase digestion, because theyare hybridized to hTRT mRNA. The non-complementary sequences aredigested away from the probe in the presence of RNase and targetcomplementary nucleic acid.

Two RNAse protection probes are described for illustrative purposes;either can be used in the assay. The probes differ in their sequencescomplementary to hTRT, but contain identical non-complementarysequences, in this embodiment, derived from the SV40 late mRNA leadersequence. From 5′-3′, one probe is comprised of 33 nucleotides ofnon-complementary sequence and 194 nucleotides of sequence complementaryto hTRT nucleotides 2513-2707 for a full length probe size of 227nucleotides. From 5′-3′, the second probe is comprised of 33 nucleotidesof non-complementary sequence and 198 nucleotides of sequencecomplementary to hTRT nucleotides 2837-3035 for a full length probe sizeof 231 nucleotides. To conduct the assay, either probe can be hybridizedto RNA, i.e., polyA+ RNA, from a test sample, and T1 ribonuclease andRNase A are then added. After digestion, probe RNA is purified andanalyzed by gel electrophoresis. Detection of a 194 nucleotide fragmentof the 227 nucleotide probe or a 198 nucleotide fragment of the 231nucleotide probe is indicative of hTRT mRNA in the sample.

The illustrative RNAse protection probes described in this example canbe generated by in vitro transcription using T7 RNA polymerase.Radioactive or otherwise labeled ribonucleotides can be included forsynthesis of labeled probes. The templates for the in vitrotranscription reaction to produce the RNA probes are PCR products. Theseillustrative probes can be synthesized using T7 polymerase following PCRamplification of pGRN121 DNA using primers that span the correspondingcomplementary region of the hTRT gene or mRNA. In addition, thedownstream primer contains T7 RNA polymerase promoter sequences and thenon-complementary sequences.

For generation of the first RNAse protection probe, the PCR product fromthe following primer pair (T701 and reverse01) is used:

T701 5′-GGGAGATCT TAATACGACTCACTATAG ATTCA GGCCATGGTGCTGCGCCGGC TGTCAGGCTCCC ACGACGTAGT CCATGTTCAC-3′ (SEQ ID NO:624); and reverse015′-GGGTCTAGAT CCGGAAGAGTGT CTGGAGCAAG-3′ (SEQ ID NO:625).

For generation of the second RNase protection probe, the PCR productfrom the following primer pair (T702 and reverse02) is used:

T702 5′-GGGAGATCT TAATACGACTCACTATAG ATTCA GGCCATGGTGCTGCGCCGGC TGTCAGGGCG GCCTTCTGGA CCACGGCATA CC-3′ (SEQ ID NO:626); and reverse02 5′-GGTCTAGA CGATATCC ACAGGGCCTG GCGC-3′ (SEQ ID NO:672).

Example 12 Construction of a Phylogenetic Tree Comparing hTRT and OtherReverse Transcriptases

A phylogenetic tree (FIG. 6) was constructed by comparison of the sevenRT domains defined by Xiong and Eickbush (1990, EMBO J. 9:3353). Aftersequence alignment of motifs 1, 2, and A-E from 4 TRTs, 67 RTs, and 3RNA polymerases, the tree was constructed using the NJ (NeighborJoining) method (Saitou and Nei, 1987, Mol. Biol. Evol. 4:406). Elementsfrom the same class that are located on the same branch of the tree aresimplified as a box. The length of each box corresponds to the mostdivergent element within that box.

The TRTs appear to be more closely related to RTs associated with msDNA,group II introns, and non-LTR (Long Terminal Repeat) retrotransposonsthan to the LTR-retrotransposon and viral RTs. The relationship of thetelomerase RTs to the non-LTR branch of retroelements is intriguing,given that these latter elements have replaced telomerase for telomeremaintenance in Drosophila. However, the most striking finding is thatthe TRTs form a discrete subgroup, almost as closely related to theRNA-dependent RNA polymerases of plus-stranded RNA viruses such aspoliovirus as to any of the previously known RTs. Considering that thefour telomerase genes come from evolutionarily distantorganisms—protozoan, fungi, and mammal—this separate grouping cannot beexplained by lack of phylogenetic diversity in the data set. Instead,this deep bifurcation suggests that the telomerase RTs are an ancientgroup, perhaps originating with the first eukaryote.

GenBank protein identification or accession numbers used in thephylogenetic analysis were: msDNAs (94535, 134069, 134074, 134075,134078), group II introns (483039, 101880, 1332208, 1334433, 1334435,133345, 1353081), mitochondrial plasmid/RTL (903835, 134084), non-LTRretrotransposons (140023, 84806, 103221, 103353, 134083, 435415, 103015,1335673, 85020, 141475, 106903, 130402, U0551, 903695, 940390, 2055276,L08889), LTR retrotransposons (74599, 85105, 130582, 99712, 83589,84126, 479443, 224319, 130398, 130583, 1335652, 173088, 226407, 101042,1078824), hepadnaviruses (I 18876, 1706510, 118894), caulimoviruses(331554, 130600, 130593, 93553), retroviruses (130601, 325465, 74601,130587, 130671, 130607, 130629, 130589, 130631, 1346746, 130651, 130635,1780973, 130646). Alignment was analyzed using ClustalW 1.5 [J. D.Thompson, D. G. Higgins, T. J. Gibson, Nucleic Acids Res. 22, 4673(1994)] and PHYLIP 3.5 [J. Felsenstein, Cladisfics 5, 164 (1989)].

Example 13 Transfection of Cultured Human Fibroblasts (BJ) with ControlPlasmid and Plasmid Encoding hTRT

This example demonstrates that expression of recombinant hTRT protein ina mammalian cell results in the generation of an active telomerase.

Subconfluent BJ fibroblasts were trypsinized and resuspended in freshmedium (DMEM/199 containing 10% Fetal Calf Serum) at a concentration of4×10⁶ cells/ml. The cells were transfected using electroporation withthe BioRad Gene Pulser™ electroporator. Optionally, one may alsotransfect cells using Superfect™ reagent (Qiagen) in accordance with themanufacturer's instructions. For electroporation, 500 μl of the cellsuspension were placed in an electroporation cuvette (BioRad, 0.4 cmelectrode gap). Plasmid DNA (2 μg) was added to the cuvettes and thesuspension was gently mixed and incubated on ice for 5 minutes. Thecontrol plasmid (pBBS212) contained no insert behind the MPSV promoterand the experimental plasmid (pGRN133) expressed hTRT from the MPSVpromoter. The cells were electroporated at 300 Volts and 960 μFD. Afterthe pulse was delivered, the cuvettes were placed on ice forapproximately 5 minutes prior to plating on 100 mm tissue culture dishesin medium. After 6 hours, the medium was replaced with fresh medium. 72hours after the transfection, the cells were trypsinized, washed oncewith PBS, pelleted and stored frozen at −80° C. Cell extracts wereprepared at a concentration of 25,000 cells/μl by a modified detergentlysis method (see Bodnar et al., 1996, Exp. Cell Res. 228:58; Kim etal., 1994, Science 266:2011, and as described in patents andpublications relating to the TRAP assay, supra) and telomerase activityin the cell extracts was determined using a modified PCR-based TRAPassay (Kim et al., 1994, Bodnar et al., 1996). Briefly, 5×10⁴ cellequivalents were used in the telomerase primer extension portion of thereaction. While the extract is typically taken directly from thetelomerase extension reaction to the PCR amplification, one may alsoextract once with phenol/chloroform and once with chloroform prior tothe PCR amplification. One-fifth of the material was used in the PCRamplification portion of the TRAP reaction (approximately 10,000 cellequivalents). Onehalf of the TRAP reaction was loaded onto the gel foranalysis, such that each lane in FIG. 25 represents reaction productsfrom 5,000 cell equivalents. Extracts from cells transfected withpGRN133 were positive for telomerase activity while extracts fromuntransfected (not shown) or control plasmid transfected cells showed notelomerase activity. Similar experiments using RPE cells gave the sameresult.

Reconstitution in BJ cells was also carried out using other hTRTconstructs (i.e., pGRN145, pGRN155 and pGRN138). Reconstitution usingthese constructs appeared to result in more telomerase activity than inthe pGRN133 transfected cells.

The highest level of telomerase activity was achieved using pGRN155. Asdiscussed supra, pGRN155 is a vector containing the adenovirus majorlate promoter as a controlling element for the expression of hTRT andwas shown to reconstitute telomerase activity when transfected into BJcells.

Notably, when reconstitution using the hTRT-GFP fusion protein pGRN138(which localizes to the nucleus, see Example 15, infra) was performedeither in vitro (see Example 7) or in vivo (transfection into BJ cells)telomerase activity resulted. By transfection into BJ cells, forexample, as described supra, telomerase activity was comparable to thatresulting from reconstitution in vitro using pGRN133 or pGRN145.

Similar results were obtained upon transfection of normal human retinalpigmented epithelial (RPE) with the hTRT expression vectors of theinvention. The senescence of RPE cells is believed to contribute to orcause the disease of age-related macular degeneration. RPE cells treatedin accordance with the methods of the invention using the hTRTexpression vectors of the invention should exhibit delayed senescence,as compared to untreated cells, and so be useful in transplantationtherapies to treat or prevent age-related macular degeneration.

Example 14 Promoter Reporter Construct

This example describes the construction of plasmids in which reportergenes are operably linked to hTRT upstream sequences containing promoterelements. The vectors have numerous uses, including identification ofcis and trans transcriptional regulatory factors in vivo and forscreening of agents capable of modulating (e.g., activating orinhibiting) hTRT expression (e.g., drug screening). Although a number ofreporters may be used (e.g., firefly luciferase, β-glucuronidase,β-galactosidase, chloramphenicol acetyl transferase, and GFP and thelike), the human secreted alkaline phosphatase (SEAP; CloneTech) wasused for initial experiments. The SEAP reporter gene encodes a truncatedform of the placental enzyme which lacks the membrane anchoring domain,thereby allowing the protein to be secreted efficiently from transfectedcells. Levels of SEAP activity detected in the culture medium have beenshown to be directly proportional to changes in intracellularconcentrations of SEAP mRNA and protein (Berger et al., 1988, Gene 66:1;Cullen et al., 1992, Meth. Enzymol. 216:362).

Four constructs (pGRN148, pGRN150, “pSEAP2 basic” (no promotersequences=negative control) and “pSEAP2 control” (contains the SV40early promoter and enhancer) were transfected in triplicate into mortaland immortal cells.

Plasmid pGRN148 was constructed as illustrated in FIG. 9. Briefly, aBgl2-Eco47III fragment from pGRN144 was digested and cloned into theBglII-NruI site of pSeap2Basic (Clontech, San Diego, Calif.). A secondreporter-promoter, plasmid pGRN150, includes sequences from the hTRTintron described in Example 3, to employ regulatory sequences that maybe present in the intron. The initiating Met is mutated to Leu, so thatthe second ATG following the promoter region will be the initiating ATGof the SEAP ORF.

The pGRN148 and pGRN150 constructs (which include the hTRT promoter)were transfected into mortal (BJ cells) and immortal (293) cells. Alltransfections were done in parallel with two control plasmids: onenegative control plasmid (pSEAP basic) and one positive control plasmid(pSEAP control which contains the SV40 early promoter and the SV40enhancer).

In immortal cells, pGRN148 and pGRN150 constructs appear to drive SEAPexpression as efficiently as the pSEAP2 positive control (containing theSV40 early promoter and enhancer). In contrast, in mortal cells only thepSEAP2 control gave detectable activity. These results indicate that, asexpected, hTRT promoter sequences are active in tumor cells but not inmortal cells.

Similar results were obtained using another normal cell line (RPE, orretinal pigmental epithelial cells). In RPE cells transfected withpGRN150 (containing 2.2 KB of upstream genomic sequence), the hTRTpromoter region was inactive while the pSEAP2 control plasmid wasactive.

As noted supra, plasmids in which reporter genes are operably linked tohTRT upstream sequences containing promoter elements are extremelyuseful for identification and screening of telomerase activitymodulatory agents, using both transient and stable transfectiontechniques. In one approach, for example, stable transformants ofpGRN148 are made in telomerase negative and telomerase positive cells bycotransfection with a eukaryotic selectable marker (such as neo)according to Ausubel et al., 1997, supra. The resulting cell lines areused for screening of putative telomerase modulatory agents, forexample, by comparing hTRT-promoter-driven expression in the presenceand absence of a test compound.

The promoter-reporter (and other) vectors of the invention are also usedto identify trans- and cis-acting transcriptional and translationalregulatory elements. Examples of cis-acting transcriptional regulatoryelements include promoters and enhancers of the telomerase gene. Theidentification and isolation of cis- and trans-acting regulatory agentsprovide for further methods and reagents for identifying agents thatmodulate transcription and translation of telomerase.

To identify sequences or elements that play a role in hTRT expression,expression was tested using promoter-reporter constructs with varyingamounts of the upstream region (5′ to the transcription initiation site)of the hTRT gene. Experiments were conducted using pGRN 150 [whichcontains approximately 2405 bp of genomic sequence upstream of the most5′ nucleotide present in the hTRT cDNA], pGRN 176 [which containsapproximately 186 bp of genomic sequence upstream of the most 5′nucleotide present in the hTRT cDNA] and pGRN 175 [which containsapproximately 77 bp of genomic sequence upstream of the most 5′nucleotide present in the hTRT cDNA]. The following sequence is presentin pGRN 176 but not pGRN 175:5′-GTGGCGGAGGGACTGGGGACCCGGGCACCGGTCCTGCCCCTTCACCTTCCAGCTCCGCCTCGTCCGCGCGGAACCCCGCCCCGTCCCGAACCCTTCCCGGGTCCCCGGCCCAGCCCCTTCCGGG-3′(SEQ ID NO:726).

When transfected into mortal cells (RPE and BJ), the pGRN 175 promoterwas active, while the pGRN 176 and pGRN 150 promoters were not active.These results demonstrate that the approximately 120 basepair regionpresent in pGRN 176 but not pGRN 175 includes sequences that play a rolein the mortal-cell specific repression of hTRT gene expression isachieved. It will be recognized that less than the entire approximately120 basepair sequence may be required for this effect, and that othersequences not in the approximately 120 base pair region may also play arole (independently or in combination with the approximately 120 basepair region) in regulation of hTRT expression. Thus, the approximately120 base pair region includes all or part of one or more cis-actingelements.

Without intending to be bound by any particular mechanism, theapproximately 120 base pair sequence includes a binding site for arepressor (e.g., a trans acting repressor) which upon binding preventsinitiation of transcription of the hTRT gene. Such a repressor may bethe product of an anti-oncogene (e.g., a novel anti-oncogene), which canbe identified and cloned in accordance with the teachings herein and theuse of the novel reagents disclosed herein. In normal cells, repressorbinding or interaction with hTRT regulatory sequences (e.g., includingor within the approximately 120 base pair sequence) results in theabsence of hTRT protein and therefore of telomerase activity. Activationof telomerase in cancer cells can result from the loss of hTRT repressoractivity.

A number of applications of the “approximately 120 base pair region”described above will be immediately apparent upon review of thisdisclosure, including for treatment or diagnosis of telomerase relateddiseases and identification of agents with telomerase modulatoryactivity. For example, using standard techniques, the sequence may beused to identify agents or proteins (e.g. naturally occurring repressorproteins) that specifically bind to the approximately 120 base pairsequence or a subsequence thereof. In addition, synthetic or naturallyoccurring agents that increase or stabilize repression (e.g., by bindingor otherwise interacting with the sequence, by stabilizing binding by anaturally occurring repressor, or by other means) will be useful forreducing telomerase activity in a cell (e.g., for treatment ofmalignancy). Similarly, agents that reduce repression (e.g., byinhibiting repressor binding, or by other means) will be useful forincreasing telomerase expression (e.g., by controlled activation), forexample to increase the proliferative capacity of normal cells).

Example 15 Subcellular Localization of hTRT

A fusion protein having hTRT and enhanced green fluorescent protein(EGFP; Cormack et al., 1996, Gene 173:33) regions was constructed asdescribed below. The EGFP moiety provides a detectable tag or signal sothat the presence or location of the fusion protein can be easilydetermined. Because EGFP-fusion proteins localize in the correctcellular compartments, this construct may be used to determine thesubcellular location of hTRT protein.

A. Construction of pGRN138

A vector for expression of an hTRT-EGFP fusion protein in mammaliancells was constructed by placing the EcoRI insert from pGRN 124 (seeExample 6) into the EcoRI site of pEGFP-C2 (Clontech, San Diego,Calif.). The amino acid sequence of the fusion protein is providedbelow. EGFP residues are in bold, residues encoded by the 5′untranslated region of hTRT mRNA are underlined, and the hTRT proteinsequence is in normal font.

MVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSGRTQISSSSFEFAAASTQRCVLLRTWEALAPATPAMPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYWVELLRSFFYVTETTFQKNRLFFYRPSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASVTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD(SEQ ID NO:628)

Other EGFP fusion constructs can be made using partial (e.g., truncated)hTRT coding sequence and used, as described infra, to identifyactivities of particular regions of the hTRT polypeptide.

B. Nuclear Localization and Uses of pGRN138

Transfection of NIH 293 and BJ cells with pGRN138 confirmed the nuclearlocalization of recombinantly expressed hTRT. Cells were transfectedwith pGRN138 (EGFP-hTRT) and with a control construct (expressing EGFPonly). Nuclear localization of the EGFP-hTRT is apparent in both celltypes by fluorescence microscopy. As noted supra, the pGRN138 hTRT-GFPfusion protein supports reconstitution of telomerase activity in both anin vitro transcription translation system and in vivo when transfectedinto BJ cells.

The hTRT-EGFP fusion proteins (or similar detectable fusion proteins)can be used in a variety of applications. For example, the fusionconstruct described in this example, or a construct of EGFP and atruncated form of hTRT, can be used to assess the ability of hTRT andvariants to enter a cell nucleus and/or localize at the chromosome ends.In addition, cells stably or transiently transfected with pGRN138 areused for screening compounds to identify telomerase modulatory drugs orcompounds. Agents that interfere with nuclear localization or telomerelocalization can be identified as telomerase inhibitors. Tumor celllines stably expressing EGFP-hTRT can be useful for this purpose.Potential modulators of telomerase will be administered to thesetransfected cells and the localization of the EGFP-hTRT will beassessed. In addition, FACS or other fluorescence-based methods can beused to select cells expressing hTRT to provide homogeneous populationsfor drug screening, particularly when transient transfection of cells isemployed.

In other applications, regions of the hTRT can be mutagenized toidentify regions (e.g., residues 193-196 (PRRR; SEQ ID NO:541) and235-240 (PKRPRR; SEQ ID NO:542)) required for nuclear localization,which are targets for anti-telomerase drugs (telomerase activitymodulators). Other applications include:

use of the fusion protein as a fluorescent marker of efficient celltransfection for both transient transfection experiments and whenestablishing stable cell lines expressing EGFP-hTRT;

expression of an hTRT-EGFP fusion with mutated nuclear localizationsignals (deficient for nuclear localization) in immortal cells so thatthe hTRT mutant-EGFP scavenges all the hTR of the immortal cells,retaining it in the cytoplasm and preventing telomere maintenance; and

use as a tagged protein for immunoprecipitation.

Example 16 Effect of Mutation on Telomerase Catalytic Activity

This example describes hTRT variant proteins having altered amino acidsand altered telomerase catalytic activity. Amino acid substitutionsfollowed by functional analysis is a standard means of assessing theimportance and function of a polypeptide sequence. This exampledemonstrates that changes in the reverse transcriptase (RT) andtelomerase (T) motifs affect telomerase catalytic activity.

Conventional nomenclature is used to describe mutants: the targetresidue in the native molecule (hTRT) is identified by one-letter codeand position, and the corresponding residue in the mutant protein isindicated by one-letter code. Thus, for example, “K626A” specifies amutant in which the lysine at position 626 (i.e., in motif 1) of hTRT ischanged to an alanine.

A. Mutation of hTRT FFYxTE (SEQ ID NO:360) Motif

In initial experiments, a vector encoding an hTRT mutant protein,“F560A,” was produced in which amino acid 560 of hTRT was changed fromphenylalanine (F) to alanine (A) by site directed mutagenesis of pGRN121using standard techniques. This mutation disrupts the TRT FFYxTE (SEQ IDNO:360) motif. The resulting F560A mutant polynucleotide was shown todirect synthesis of a full length hTRT protein as assessed using acell-free reticulocyte lysate transcription/translation system in thepresence of ³⁵S-methionine.

When the mutant polypeptide was co-translated with hTR, as described inExample 7, no telomerase activity was detected as observed by TRAP using20 cycles of PCR, while a control hTRT/hTR cotranslation didreconstitute activity. With 30 cycles of PCR in the TRAP assay,telomerase activity was observable with the mutant hTRT, but wasconsiderably lower than the control (wild-type) hTRT.

B. Additional Site-Directed Mutagenesis of hTRT Amino Acid Residues

Conserved amino acids in six RT motifs were changed to alanine usingstandard site directed mutagenesis techniques (see, e.g., Ausubel,supra) to assess their contribution to catalytic activity. The mutantswere assayed using IVR telomerase using the two step conventional/TRAPassay detailed in example 7.

The K626A (motif 1), R631A (motif 2), D712A (motif A), Y717A (motif A),D868A (motif C) mutants had greatly reduced or undetectable telomeraseactivity (<1% of wild-type), while the Q833A (motif B) and G932A (motifE) mutants exhibited low/intermediate levels of activity (<10% ofwild-type). Two mutations outside the RT motifs, R688A and D897A, hadactivity equivalent to wild type hTRT. These results were consistentwith analogous mutations in reverse transcriptases (Joyce et al., 1994,Ann. Rev. Biochem. 63:777) and are similar to results obtained withEst2p (see Lingner, 1997, Science 276:561). The experiments identifyresidues in the RT motifs critical and not critical for enzymaticactivity and demonstrate that hTRT is the catalytic protein of humantelomerase. The mutations provide variant hTRT polypeptides that haveutility, e.g., as dominant/negative regulators of telomerase activity.

Amino acid alignment of the known TRTs identified a telomerase-specificmotif, motif T (see supra). To determine the catalytic role of thismotif in hTRT, a six amino acid deletion in this motif (Δ560-565;FFYxTE; SEQ ID NO:360), was constructed using standard site directedmutagenesis techniques (Ausubel, supra). The deletion was assayed usingIVR telomerase using the two step conventional/TRAP assay detailed inExample 7. The Δ560-565 mutant had no observable telomerase activityafter 25 cycles of PCR whereas wild type hTRT IVR telomerase produced astrong signal. Each amino acid in each residue in motif T was examinedindependently in a similar manner; mutants F560A, Y562A, T564A, andE565A retained intermediate levels of telomerase activity, while acontrol mutant, F487A, had minimal affect on activity. Notably, mutantF561A had greatly reduced or undetectable telomerase activity, whileactivity was fully restored in its “revertant”, F561A561F.F561A561Fchanges the mutated position back to its originalphenylalanine. This is a control that demonstrates that no other aminoacid changes occurred to the plasmid that could account for thedecreased activity observed. Thus, the T motif is the first non-RT motifshown to be absolutely required for telomerase activity.

Motif T can be used for identification of TRTs from other organisms andhTRT proteins comprising variants of this motif can be used as adominant/negative regulator of telomerase activity. Unlike most otherRTs, telomerase stably associates with and processively copies a smallportion of a single RNA (ie. hTR), thus motif T can be involved inmediating hTR binding, the processivity of the reaction, or otherfunctions unique to the telomerase RT.

In other experiments, it was observed that the deletion variant encodedby pro90hTRT described herein, did not reconstitute telomerase activitywhen co-synthesized with hTR, as measured using a modified TRAP assay(Autexier et al., 1996, EMBO Journal 15:5928, which is incorporatedherein by reference).

Example 17 Screening for Telomerase Activity Modulators UsingRecombinantly Expressed Telomerase Components

This example describes the use of in vitro reconstituted telomerase forscreening and identifying telomerase activity modulators. The assaydescribed is easily adapted to high-through-put methods (e.g., usingmultiple well plates and/or robotic systems). Numerous variations on thesteps of the assay will be apparent to one of skill in the art afterreview of this disclosure.

Recombinant clones for telomerase components (e.g., hTRT and hTR) aretranscribed and translated (hTRT only) in an in vitro reaction asfollows and as described in Example 7 supra, using the TNT7 T7 CoupledReticulocyte lysate system (Promega), which is described in U.S. Pat.No. 5,324,637, following the manufacturer's instructions:

Reagent Amount per reaction (μL) TNT Rabbit Reticulocyte lysate 25  TNTreaction buffer 2 TNT T7 RNA Pol. 1 AA mixture (complete) 1 Prime RNaseinhibitor 1 Nuclease-free water 16  Xba1 cut pGRN121 [hTRT] (0.5 μg) 2Fsp1 cut pGRN164 [hTR] (0.5 μg) 2

The reaction is incubated at 30° C. for 2 hours. The product is thenpurified on an ultrafree-MC DEAE filter (Millipore).

The recombinant telomerase product (IVRP) is assayed in the presence andabsence of multiple concentrations of test compounds which aresolubilized in DMSO (e.g. 10 μM-100 μM). Test compounds are preincubatedin a total volume of 25 μL for 30 minutes at room temperature in thepresence of 2.5 μL IVRP, 2.5% DMSO, and 1×TRAP Buffer (20 mM Tris-HCl,pH 8.3, 1.5 mM MgCl₂, 63 mM KCl, 0.05% Tween20, 1.0 mM EGTA, 0.1 mg/mlBovine serum albumin). Following the preincubation, 25 μL of the TRAPassay reaction mixture is added to each sample. The TRAP assay reactionmixture is composed of 1×TRAP buffer, 50 μL dNTP, 2.0 μg/ml primer ACX,4 μg/ml primer U2, 0.8 attomol/ml TSU2, 2 units/50 μl Taq polymerase(Perkin Elmer), and 2 μg/ml [³²P]5′end-labeled primer TS (3000 Ci/mmol).The reaction tubes are then placed in the PCR thermocycler (MJ Research)and PCR is performed as follows: 60 min at 30° C., 20 cycles of {30 secat 94° C., 30 sec. at 60° C., 30 sec. at 72° C.}, 1 min at 72° C., cooldown to 10° C. The TRAP assay is described, as noted supra, in U.S. Pat.No. 5,629,154. The primers and substrate used have the sequences: TSPrimer (5′-AATCCGTCGAGCAGAGTT-3′; SEQ ID NO:629); ACX Primer(5′-GCGCGG[CTTACC]₃CTAACC-3′; SEQ ID NO:630); U2 primer(5′-ATCGCTTCTCGGCCTTTT-3′; SEQ ID NO:631); TSU2(5′-AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3′; SEQ ID NO:632).

After completion of the PCR step, 4 μl of 10×loading buffer containingbromophenol blue is added to each reaction tube and products (20 μl) arerun on a 12.5% non-denaturing PAGE in 0.5×TBE at 400 V. The completedgel is subsequently dried and the TRAP products are visualized byPhosphorimager or by autoradiography. The telomerase activity in thepresence of the test compound is measured by comparing the incorporationof label in reaction product to a parallel reaction lacking the agent.

The following clones described in the Examples have been deposited withthe American Type Culture Collection, Rockville, Md. 20852, USA:

Lambda phage λ25-1.1 ATCC accession number 209024

pGRN121 ATCC accession number 209016

Lambda phage λGΦ5 ATCC accession number 98505

The present invention provides novel methods and materials relating tohTRT and diagnosis and treatment of telomerase-related diseases. Whilespecific examples have been provided, the above description isillustrative and not restrictive. Many variations of the invention willbecome apparent to those of skill in the art upon review of thisspecification. The scope of the invention should, therefore, bedetermined not with reference to the above description, but insteadshould be determined with reference to the appended claims along withtheir full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted.

SEQUENCE LISTING The patent contains a lengthy “Sequence Listing”section. A copy of the “Sequence Listing” is available in electronicform from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=06617110B1). An electroniccopy of the “Sequence Listing” will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. A method of drug screening or validation of adrug, comprising: a) obtaining a drug or drug candidate, b) obtaining acultured mammalian cell comprising a nucleic acid sequence that encodesa telomerase reverse transcriptase protein, variant, or fragment,wherein said variant, or fragment, has telomerase catalytic activitywhen complexed with a telomerase RNA, wherein the nucleic acid sequencehybridizes under stringent conditions to a polynucleotide having asequence complementary to SEQ ID NO: 1, and wherein the expression ofthe protein, variant, or fragment in the cell increases the number ofdivisions the cell can undergo before senescence; c) administering thedrug or drug candidate to the cultured cell; and d) determining if thedrug or candidate has an effect on the cell.
 2. The method of claim 1,wherein the cell is a human cell.
 3. The method cell of claim 2, whereinthe cell further comprises a selectable marker gene.
 4. The method ofclaim 2, wherein the nucleic acid comprises a constitutive promoter. 5.The method of claim 2, wherein the nucleic acid comprises an induciblepromoter.
 6. The method of claim 2, wherein the cell is a liver cell. 7.The method of claim 6, wherein the cell is a hepatocyte.
 8. The methodof claim 2, wherein the cell is a nerve cell.
 9. The method of claim 8,wherein the cell is a glial cell, astrocyte, or oligodendrocyte.
 10. Themethod of claim 8, wherein the cell is a neuron of the central nervoussystem.
 11. The method of claim 10, wherein the cell is a cholinergic oradrenergic cell.
 12. The method of claim 2, wherein the cell is aretinal pigmented epithelial cell.
 13. The method of claim 2, whereinthe cell is a contractile cell.
 14. The method of claim 13, wherein thecell is a heart muscle cell or smooth muscle cell.
 15. The method ofclaim 2, wherein the cell is a fat cell.
 16. The method of claim 2,wherein the cell is a fibroblast.
 17. The method of claim 2, wherein thecell is a vascular endothelial cell.
 18. The method of claim 2, whereinthe cell is a hormone secreting cell.
 19. The method of claim 18,wherein the cell secretes insulin or glucagon.
 20. The method of claim18, wherein the cell is a pituitary cell, thyroid hormone secretingcell, or adrenal cell.
 21. The method of claim 2, wherein the cell is afat storing cell.
 22. The method of claim 2, wherein the cell is anepithelial or mucosal cell.
 23. The method of claim 22, wherein the cellis an oral cavity cell, stomach cell, or intestinal cell.
 24. The methodof claim 22, wherein the cell is a mammary gland, uterus, or prostatecell.
 25. The method of claim 22, wherein the cell is an air spaceepithelial cell or the lung.
 26. The method of claim 2, wherein the cellis a tubular cell of the kidney.
 27. The method of claim 2, wherein thecell is a blood cell or a cell of the immune system,
 28. The method ofclaim 27, wherein the cell is a T or B lymphocyte.
 29. The method ofclaim 27, wherein the cell is a mast cell or eosinophil.
 30. The methodof claim 27, wherein the cell is a monocyte or macrophage.
 31. Themethod of claim 2, wherein the cell is an osteoblast, osteocyte, orosteoclast.
 32. The method of claim 2, wherein the cell is a chondrocyteor synovial cell.
 33. The method of claim 2, wherein the cell is a stemcell.
 34. The method of claim 33, wherein the cell is an embryonic stemcell.
 35. The method of claim 33, wherein the cell is an embryonic germcell.
 36. The method of claim 33, wherein the cell is an adult stemcell.
 37. The method of claim 2, wherein the nucleic acid encodes afull-length, naturally occurring human telomerase reverse transcriptase.38. The method of claim 2, wherein the nucleic acid encodes afull-length, naturally occurring human telomerase reverse transcriptasehaving the amino acid sequence of SEQ ID NO:
 2. 39. The method of claim1, comprising determining whether the drug or drug candidate is lethalto the cell.